Yeast membrane protein expression system and its application in drug screening

ABSTRACT

The invention relates to an in vitro cell based expression system for overexpressing heterologous pump proteins associated with drug resistance into the membrane of the host cell for drug screening applications.

FIELD OF THE INVENTION

This invention relates to a protein expression system, particularlyalthough by no means exclusively, to a plasma membrane proteinexpression system, and the application of this system in understandingthe basic biology of membrane bound proteins and in drug discovery.

BACKGROUND

Proteins located in the plasma membrane or surface membranes of targetcells are amongst the most prominent, accessible and attractive sitesfor intervention with small molecule drugs for pharmaceutical andagrochemical purposes. For example, drugs such as ouabain and thecardiac glycosides are effective therapeutics in the treatment of heartdisease because of their activity against isoforms of the membraneprotein Na⁺,K⁺-ATPase of mammalian cells (Schwartz A, et al, 1982).

Individual membrane proteins of interest that are located at the cellsurface may be constitutively expressed cellular components found in ahost or a pathogenic organism. Alternatively, the expression of theseproteins may also be affected by mutation or by interactions betweensuch cells and other organisms. These membrane proteins includetransporters, channels, receptors and enzymes plus proteins withstructural, regulatory or unknown roles. Various members of theseclasses of proteins are known to affect the growth, viability, andfunctional capacity of host organisms, tissues or cells. In particular,several classes of membrane proteins are known to be involved in drugresistance. These include the drug efflux pump proteins which act toincrease the efflux of particular drugs, such as antibiotics and otherxenobiotics, from the inside of a cell to the outside. This activitylowers the concentration of the drug at the intracellular target site tolevels which are no longer effective. Yeast cell expression systems fortesting drugs that inhibit drug efflux pump proteins are known.Decottignies et al 1998 describes a number of strains of Saccharomycescerevisiea in which varying endogenous drug efflux pump proteins (ABCtransporter proteins) have been deleted and a further endogenousmembrane protein overexpressed in the cell membrane. Such a system alsoemploys the use of regulators which aid in this overexpression. Examplesof such regulators are described in Carjaval et al (1997). However, sucha system is restrictive in its application as it may be speciesspecific, ie it may only identify potential drugs useful in inhibitingdrug resistance in Saccharomyces cerevisiea.

As the problem of drug resistance is widely found in all fauna andflora, and not just in yeast, there exists a need to develop a simple invitro cell based membrane protein expression system for testingpotential inhibitors of drug efflux pump proteins, as well as othermembrane proteins associated with drug resistance, from differentspecies.

In addition, as the number of potential test compounds, located mainlyin compound libraries, is increasing in both size and complexity, thereis a need for such a simple in vitro, cell based membrane proteinexpression system to screen for agonists or antagonists of putativemembrane protein drug targets from a broad range of species and whichcan be adapted for high throughput formats.

It is an object of the present invention to go some way towardsproviding for these needs and/or to provide the public with a usefulchoice.

SUMMARY OF INVENTION

The present invention provides a protein expression system comprising:

-   -   i) a host yeast cell; and    -   ii) a vector containing the coding sequence of a target        heterologous membrane protein, said sequence being under the        control of a promoter which, upon transformation of said host        cell and chromosomal integration, causes over-expression of the        functional target protein in the membrane of the host cell.

The host yeast cell may comprise a strain of the genus Saccharomyces.

The host yeast cell may comprise a mutant strain deficient in one ormore naturally occurring membrane proteins, such as drug efflux pumps,so that the target protein expressed in the membrane of the host cell,is relatively prominent and accessible for drug screening applications.The preferred yeast strain is Saccharomyces cerevisiea AD1-8u⁻.

In some applications, the host cell may contain a mutation that leads tothe formation of secretory vesicles whose ability to fuse normally withthe plasma membrane is temperature sensitive. A preferred mutated strainis the sec6-4 mutant of the AD1-8u⁻ strain.

The coding sequence of the target heterologous protein may beincorporated into the host cell in a defined location in the genome suchas downstream of an endogenous promoter.

The coding sequence of the target heterologous membrane protein maycomprise the entire natural coding sequence of the target protein, or afunctional fragment or variant thereof which, upon transformation andexpression, will produce a functional membrane protein with a detectablephenotype.

The target heterologous membrane protein of the invention may comprise adrug efflux pump protein such as those involved in multidrug resistancein fungi, but may also include other molecules such as theP-glycoprotein, the cystic fibrosis transmembrane conductance regulatorand other human, animal, plant and microbial plasma membrane proteinsthat play a role in the conferral of resistance or sensitivity toxenobiotics, the etiology of disease or the modulation of physiology,growth and development.

Preferably, the target membrane protein is a drug efflux pump proteinand the candidate compound is an efflux pump inhibitor.

The vector used to integrate the coding sequence of the targetheterologous membrane protein is preferably a plasmid vector whichcontains elements which allow replication in E. coli. The vector mayalso include a transcription terminator that is functional in the hostcell. In addition, the vector may include a marker that confers aselectable phenotype on the cells after transformation. The promoter isselected from the group of promoters comprising constitutive S.cerevisiae PDR5 and PMA1 promoters, copper controllable CTR3, glucoseinducible ADH1 and PGK promoters, the galactose inducible GAL promoter,the doxycycline controllable bacterial tet0 promoter and thetet0::ScHOP1 controllable cassette. The preferred promoter is PDR5. Thepreferred vector is pABC3.

The yeast host strain may further comprise a mutated transcriptionalregulator coding sequence that causes overexpression of the targetcoding sequence leading to abundant expression of the target protein inthe membrane of the host cell. The mutated transcriptional mutator maybe Pdrl-3p.

The present invention further provides a method of screening for drugsuseful as a pharmaceutical or agrochemical comprising the steps of:

-   -   i) transforming the chromosomal DNA of a host yeast cell with        DNA comprising the coding sequence of a target heterologous        membrane protein, said sequence being under the control of a        host promoter leading to over-expression of the functional        target protein in the membrane of the host cell;    -   ii) introducing at least one candidate compound to said host        cell environment or the environment of a plasma membrane        fraction derived from the transformed host strain; and    -   iii) measuring the effect, if any, of the candidate compound on        the host cell growth and/or viability and/or specific        biochemical or physiological functions mediated by the target        membrane protein; and/or measuring the binding of the candidate        compound to the target membrane protein.

The target heterologous membrane protein of the invention may comprise adrug efflux pump protein such as those involved in multidrug resistancein fungi, but may also include other molecules such as theP-glycoprotein, the cystic fibrosis transmembrane conductance regulatorand other human, animal, plant and microbial plasma membrane proteinsthat play a role in the conferral of resistance or sensitivity toxenobiotics, the etiology of disease or the modulation of physiology,growth and development.

Preferably, the target membrane protein is a drug efflux pump proteinand the candidate compound is an efflux pump inhibitor.

Preferably, the host yeast cell is of the genus Saccharomyces, and mostpreferably the host cell is a Saccharomyces cell which has beengenetically altered to be depleted in one or more natural membraneproteins. A suitable host cell is the Saccharomyces cerevisiae AD1-8u⁻strain. In another embodiment a suitable host cell may be a sec6-4mutant of the AD1-8u⁻ strain. In a further embodiment the host strainmay be a derivative of the AD1-8u⁻ strain modified to select for a novelphenotype, such as prototrophy, an auxotrophic requirement or drugsensitivity.

A transformation cassette derived from a plasmid vector may be used totransform the chromosomal DNA of the host cell. The vector may containelements which allow replication in Escherichia coli, plus a promotersuch as a Sacharomyces cerevisiae promoter and more preferably the PDR5promoter. Activity of the Saccharomyces promoter is preferably under theadditional control of a mutated transcriptional regulator causingover-expression of the target coding sequence, leading to abnormalexpression of the target protein in the membrane of the host cell. Themutated transcriptional regulator Pdr1-3p is preferred and is located inthe genome of the host cell. In some applications, a transcriptionalterminator that is functional in yeast may be included in the vector.Either the natural terminator of the gene encoding the membrane proteinor the yeast PGK1 terminator is preferred. In other applicationsimmunological, affinity or fluorescent tags may be included in thevector. In some further applications, a selectable marker may also beincluded in the vector such as S. cerevisiae URA3 marker. In otherapplications a S. cerevisiae centromere or autonomously replicatingsequence might be included in the vector. The vector is preferablypABC3.

Compounds which are identified as useful bioactives, pharmaceuticals oragrochemicals using the method and system of the invention also formpart of the present invention. These may include compounds obtained fromcompound libraries, such as NK20 as defined below.

The method and system of the present invention may also find applicationfor the over-expression of yeast and heterologous target membraneproteins for the purposes of physiological study, biochemical analysis,enzyme purification and structural analysis of said target membraneproteins. Purified membrane proteins produced by the method and systemof the invention also form part of the present invention.

In a further embodiment, the present invention provides a kit forscreening for drugs useful as a pharmaceutical or agrochemicalcomprising the protein expression system of the present inventiontogether with suitable instructions.

In a further form of the invention the target membrane protein may berequired for viability or virulence of a pathogen or the progression ofa disease. For example, the target protein may be required for theattachment or uptake of viruses or other pathogens. In such cases, theeffect, if any, of a compound on the function of the target membraneprotein may be measured.

Although the invention is broadly as defined above, it is not limitedthereto and also includes embodiments of which the following descriptionprovides non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described with reference to thefigures of the accompanying drawings in which:

FIG. 1 (A) shows the structure of the pSK-PDR5PPUS plasmid and (B) showsthe structure of the derivative plasmid pABC3.

FIG. 2. (A) shows the construction of plasmid pKEN1002 and theintegration of the C. albicans CDR1 gene at the chromosomal PDR5 locusof S. cerevisiae AD1-8u⁻.

FIG. 3. shows confirmation of the integration of a single copy of C.albicans CDR1 at the PDR5 locus in S. cerevisiae AD1002. Intact orendonuclease-restricted AD1002 genomic DNA was electrophoresed in anagarose gel, vacuum blotted onto nylon membrane and hybridised with[α-³²P]dCTP-labeled C. albicans CDR1 probe under high stringencyconditions.

FIG. 4. shows expression of C. albicans CDR1 mRNA and Cdr1p in S.cerevisiae AD1002. (A) RNA obtained from the parental strain AD1-8u⁻,AD1-8u⁻ transformed with linearised plasmid pSK-PDR5PPUS, or AD1002 washybridized with a mixture of [α-³²P]dCTP-labeled C. albicans CDR1 and S.cerevisiae PMA1 (control) probes. The lower panel shows a portion of theethidium bromide stained agarose gel before vacuum blotting. (B) Plasmamembrane proteins separated through 8% polyacrylamide gel and stainedwith Coomassie blue. (C) Plasma membrane proteins separated through 8%polyacrylamide gel were electroblotted onto nitrocellulose and incubatedwith anti-C. albicans Cdr1p antibodies. Antibodies were detected using ahorseradish peroxidase-IgG complex.

FIG. 5. shows the sensitivity of S. cerevisiae AD1002, expressing Cdr1p,to various drugs and chemicals. S. cerevisiae AD1-8u⁻ (CDR1−) or AD1002(CDR1+) cells (5×10⁵) seeded in top agar on CSM agar plates (containinguracil for AD1-8u⁻) were exposed to drugs or chemicals applied to filterdisks and incubated at 30° C. for 48 h. The sensitivity of AD1002 todrugs or chemicals was unaffected by supplementing the medium withuridine (0.02% w/v). Amounts of individual drugs applied to disks aregiven in the Materials and methods section of Example 2, below.

FIG. 6. shows oligomycin-sensitive C. albicans Cdr1p-ATPase activity inplasma membrane fractions. Membrane fractions were isolated from S.cerevisiae AD1002 (▪) or parental AD1-8u⁻ cells (●). ATPase assays werecarried out at 30° C. for 30 min, as described in Materials and methodsfor Example 2. The oligomycin-sensitive activity was determined as thedifference in ATPase activity in the presence and absence of 20 μMoligomycin. The ATPase activity was completely sensitive to vanadate(100 μM) and insensitive to aurovertin B (20 μM). The results are themeans (±SD) of four determinations carried out on two membranepreparations.

FIG. 7 shows the protein profiles of plasma membrane proteins obtainedfrom control strains AD1-8u⁻, AD-pABC, and derivative strains AD-PDR5,AD-CDR1, AD-BENR and AD-ERG11. Samples of 30 μg of SDS solubilisedprotein were separated in an 8% polyacrylamide gel and stained withCoomassie blue. Lane 1 shows the relative migration of molecular weightmarkers. Plasma membranes were obtained from strains AD-pABC (lane 2),AD-PDR5 (lane 3), AD-CDR1 (lane 4). AD-ERG11 (lane 5), AD-BEN^(R) (lane6) and AD1-8u⁻ (lane 7).

FIG. 8 shows the sensitivity of a control S. cerevisiae strain (AD-pABC,column 1) and strains expressing Pdr5p (AD-PDR5, column 2), Cdr1p(AD-CDR1, column 3), Ben^(R)p (AD-BENR, column 4) and Erg11p (AD-ERG11,column 5), to various antifungal drugs and chemicals. The cells werespread on CSM-agar plates and exposed to the drugs or chemicals appliedto filter disks and incubated at 30° C. for 48 h. The amounts ofindividual drugs are given in the Materials and methods section ofExample 3. Rhodamine 6G and rhodamine 123 were dissolved in ethanol andthe other antifungals were dissolved in DMSO.

FIG. 9 shows the effect on the expression of Pdr5p in the AD1-8u⁻background by an SfiI locus located at nucleotides −30 to −18 and a PacIlocus located at nucleotides −11 to −4 relative to the PDR5 start codon.Plasma membrane proteins (30 μg) separated through 8% polyacrylamide gelwere stained with Coomassie blue. Lane 1 shows the relative migration ofmolecular weight markers. Plasma membranes were obtained from strainsAD1-8u⁻ (lane 2), AD-PDR5 (lane 3), AD-PDR5-SfiI/PacI (lane 4).AD-PDR5-SfiI (lane 5), AD-PDR5-PacI (lane 6) and AD1234567 (lane 7).

FIG. 10. shows the in vitro characterisation of the Pdr5p inhibitorKN20. (A) Inhibition profiles of oligomycin-sensitive Pdr5p ATPaseactivity by the purified peptidesD-NH₂-asparagine-tryptophan-tryptophan-lysine-valine-arginine-arginine-arginine-CONH₂(KNO) and its singly substituted Mtr-derivative KN20. (B) Inhibitionprofile with KN20 on the oligomycin-sensitive Cdr1p ATPase activity ofplasma membranes from AD1002 minus the oligomycin-sensitive ATPaseactivity of plasma membranes from the isogenic AD1-8u⁻ strain.

FIG. 11. shows assays which measure the chemosensitisation of AD124567cells to fluconazole by the lead compound KN20. (A) Checkerboard drugsusceptibility assay of AD124567 in the presence of the indicatedconcentrations of fluconazole and KN20. (B) Disk drug susceptibilityassay of AD124567. This assay was conducted, as described in Materialsand methods for example 4, in the absence or presence of fluconazole(120 μg/ml), and in the presence of (1) KNO (47 nanomole), (2) KN20 (47nanomole) or (0) control amounts of DMSO applied to disks

FIG. 12. shows assays which measure the chemosensitisation of AD1002 andATCC 10261 cells to fluconazole by lead compound KN20. (A) Checkerboarddrug susceptibility assay of AD1002. This assay was conducted asdescribed in Materials and methods in the presence of the indicatedconcentrations of fluconazole and KN20. (B) Disk drug susceptibilityassay of AD1002. This assay was conducted, as described in Materials andmethods for Example 4, in the absence or presence of 5 μg/mlfluconazole, with (1) KNO (12 nanomole), (2) KN20 (12 nanomole) or (0)control amounts of DMSO applied to the disks. (C) Checkerboard drugsusceptibility assay of ATCC 10261. This assay was conducted, asdescribed in Material and methods for Example 4, in the presence of theindicated concentrations of fluconazole and KN20. D indicates cell deathas measured by a failure to recover any colony forming units whensamples from microtitre plate wells were cultured on solid YPD medium.

FIG. 13 shows checkerboard drug susceptibility assays of strains (a)AD-BEN^(R) and (B) AD-ERG11. The assays were conducted, as described inMaterials and methods for Example 4, in the presence of the indicatedconcentrations of fluconazole and KN20. D indicates cell death asmeasured by a failure to recover any colony forming units when samplesfrom microtitre plate wells were cultured on solid YPD medium.

FIG. 14. shows azide-dependent fluconazole accumulation by S. cerevisiaeAD1002. [³H]Fluconazole accumulation was measured in the presence (opensymbols) or absence (closed symbols) of sodium azide (20 mM). Strainsused: AD1-8u⁻ (▪,□); AD1-8u⁻/pSK-PDR5PPUS (♦,⋄) AD1002 (●,◯). Resultsare the means ±SD of six separate determinations on two batches ofcells.

FIG. 15. shows energy-dependent rhodamine 6G efflux from strainsAD124567, AD1-8u⁻ and AD1002. De-energised cells were pre-loaded withrhodamine 6G, as described in Materials and methods for Example 4. Theefflux of rhodamine 6G at 30° C. was followed by direct measurement offluorescence in cell supernatants following glucose (2 mM) addition tocell supensions. (A) The kinetics of energy-dependent rhodamine 6Gefflux in strains AD1002 (▪) and AD1-8u⁻ (●). The fluorescence insupernatants of AD1002 cells in the absence of added glucose is alsoshown (◯). (B) The effect of KN20 concentration on the energy-dependentefflux of rhodamine 6G from AD124567 cells. The indicated concentrationsof K20 were added to AD124567 cells preloaded with rhodamine 6G, thecells were preincubated for 5 minutes at 30° C. and glucose added tocommence rhodamine efflux.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention primarily relates to a method of screeningpotentially useful drugs for animal, human and plant applicationsessentially using a system which involves the genetic construction ofplasmid vectors that, upon transformation of a suitable host, enable theheterologous over-expression, analysis and application of fullyfunctioning cell surface target membrane proteins. Preferred targetmembrane proteins are those involved in multidrug efflux, and preferredhost cells include laboratory strains of yeast that are preferably alsodepleted in endogenous membrane transporters that may carry out similarfunctions. However, this method may also be useful for screening drugswhereby the target is a protein or enzyme which carries out a cellularfunction that can lead to drug resistance or some other detectablephenotype when expression of the target is increased in a null orsuitably sensitive genetic background. Such a target might be a membraneprotein localized to the plasma membrane but it could include othermembrane bound or soluble proteins localized to other organelles orsub-cellular compartments.

The system is primarily designed to provide stable, high level,functional expression of target membrane proteins. This is preferablyachieved by engineering appropriate elements of the gene encoding thetarget membrane protein, together with a transcriptional terminator anda downstream selectable marker, into a chromosomal copy of anon-essential yeast gene which is also under the control of atranscriptional regulator. This type of construct, together with acontrol null mutant, provides a system in which potential and actualcell surface targets can be selectively and functionally over-expressedto facilitate the physiological and biochemical characterisation of suchtargets and their use for drug discovery purposes. The system alsoincludes the possibility of functionally over-expressing membrane-boundor soluble proteins localized to other organelles or sub-cellularcompartments

The yeast S. cerevisiae is the preferred yeast host cell. S. cerevisiaeprovides a valuable system for drug discovery because its genome hasbeen entirely sequenced and extensively annotated, its genetics are bothwell understood and tractable, while its ease of culture can allowcell-based assays compatible with the microtitre plate formats that areconventionally used for both manual and high throughput screening.

The present invention provides a system for over-expressing aheterologous membrane protein to a level so that said proteinconstitutes 10-20% of plasma membrane protein and is thereforesufficiently prominent to measure the effects of the test drugs. Thesuccessful high level (>10% of plasma membrane protein), heterologousexpression of plasma membrane proteins in S. cerevisiae is accomplishedfor the first time. Previous attempts at heterologous expression ofmembrane proteins in various systems has not been successful (Mahanty etal 1994; Luo et al, 1999; Mao & Scarborough, 1997 and Huang et al 1996).One reason for this may be inappropriate intracellular trafficing thatcan be affected by growth medium and growth stage (de Kerchove d'ExaerdeA, et al, 1995). Another reason may be incompatibility with endogenoussystems responsible for the correct folding of newly synthesised proteinproducts. In addition, these prior art used episomal vector basedsystems which require continuous selection and give variable results asindividual organisms in a population can carry different loads of thevector. The incorporation of a single copy of the heterologous gene intothe genome of the expressing organism as used in the present inventionprovides a defined and stable genetic load. Thus the present systemwhich achieves a stable and high level functional expression ofheterologous proteins in the plasma membrane without being compromisedby mistrafficing and misfolding, therefore provides a considerableadvantage over the prior art systems.

The present invention is thus directed to the use of S. cerevisiae andmore particularly to mutant strains thereof which are geneticallyengineered to be deficient in selected membrane proteins, therebyproviding a suitable null phenotype as a host cell in which to inducethe overexpression of the target membrane protein. As an example, thestrain AD1-8u⁻, which is deficient in 7 major ABC membrane transporters,has been identified and used as a suitable host cell in which to inducethe over-expression of a heterologous target membrane protein. TheAD1-8u⁻ strain is prepared in accordance with the teaching ofDecottignes et al, 1998, in which this strain is referred to asAD12345678. In this host cell the phenotype provides susceptibility tothe azole and triazole drugs plus a wide range of xenobiotics that usemultidrug efflux pumps of the ABC transporter class. It is alsoenvisaged that other drug susceptible or nutrient-requiring phenotypesmay be created by the selective elimination of endogenous targettransporters or enzymes in yeast or other cell types for use in thepresent invention as would be appreciated by a skilled worker;

The present invention also provides the genetic modification of theAD1-8u⁻ host strain to contain a sec6-4 mutation. Sec6-4 is atemperature sensitive mutation of S. cerevisiae which is permissive forthe fusion of secretory vesicles with the plasma membrane attemperatures up to 30° C. At the non-pemissive temperature of 37° C. themembrane fusion process is blocked and causes lethality after severalhours. As expected, AD1-8u⁻ sec6-4 cells are fluconazole sensitive, grownormally at 30° C., but fail to grow at 37° C. Transformants of thishost are therefore expected to overexpress genes encoding plasmamembrane proteins inserted into the PDR5 locus at both 30° C. and 37° C.Thus a membrane protein destined for the plasma membrane, whosesynthesis is induced by the interaction between the Pdr1-3ptranscription factor and the PDR5 promoter, should be integratednormally into the plasma membrane at 30° C. but retained in secretoryvesicles at 37° C. The resultant secretory vesicles are expected to beelectrochemically active and their constituent integral membraneproteins oriented vectorially in the membrane. For example, elements ofthe cytoplasmic catalytic and nucleotide-binding domains of ABC-typemultidrug efflux proteins should be exposed on the external face of thevesicles. Conversely, the extracellular elements of such plasma membraneproteins should project into the lumen of the secretory vesicle. Theselatter parts should only be accessible to membrane impermeant reagentsif the lipid bilayer of the secretory vesicle is disrupted. Thisinvention therefore includes the possibility of using whole cells,isolated plasma membranes and secretory vesicles to identify themolecular surface that binds membrane impermeant compounds and to assessthe electrochemical properties of targeted membrane proteins. Thefunctional hyper-expression of membrane proteins in secretory vesicles,particularly those proteins with transport and/or electrochemicalfunction, provides a new tool to evaluate the properties of suchmolecules.

The plasmid vector used in the system of the present invention, wasderived from the plasmid pSK-PDR5PPUS (FIG. 1) and includes the codingsequence of a target membrane protein whereby said sequence is under thecontrol of a promoter and/or transcriptional regulator such thatover-expression of the target protein is induced. The pdr1-3 mutation inthe S. cerevisiae Pdr1p transcriptional regulator has been shown tohyper-induce the PDR5 gene promoter and cause high-level functionalover-expression of the Pdr5p protein in yeast plasma membranes (Balzi E,et al, 1994; Carvajal E, et al, 1997; Decottignies A, et al, 1994). Thesystem of the present invention effectively replaces the coding sequenceof Pdr5p with the coding sequence of the target heterologous membraneprotein plus a transcription terminator.

The pSK-PDR5PPUS plasmid was modified to improve its performance in thecloning of coding regions and in engineering yeast strains thathyper-express these constructs under the control of the Pdr1ptranscription factor containing the pdr1-3 mutation (Pdr1-3p). FIG. 1Bshows a new pABC3 vector based on the prototypic vector pSK-PDR5PPUS(FIG. 1A). Both vectors can replicate in E. coli but not in S.cerevisiae. In pABC3, pSK-PDR5PPUS has been modified to deleterestriction sites between the HindIII and BamHI cutting sites in theoriginal multicloning site and to insert the S. cerevisiae PGK1transcriptional terminator. The pABC3 vector also includes several8-base pair restriction enzyme recognition sites (SbfI and AscIimmediately upstream of the PDR5 promoter, PacI and NotI near thejunction between the PRD5 promoter and the PGK1 terminator and FseIdownstream of the PDR5 ORF, and an EcoR1 site downstream of the URA3marker). These changes were incorporated to provide a universal yeastterminator, to aid in the production of vectors carrying alternativegenetic markers, to assist the directional cloning of PCR productscontaining coding regions and to facilitate the excision from the vectorof the PDR5-imbedded URA3-containing transformation cassette for thepurpose of replacing the chromosomal PDR5 locus with the transformationcassette. It is also envisaged that the plasmid vector pSK-PDR5PPUS orit derivatives may be cloned into yeast centromeric or multicopyvectors, for example, to allow for high level inducible expression ofmembrane proteins in accordance with the present invention.

The pdr1-3 mutation was used in the system of the present invention todrive the stable over-expression of heterologous genes inserted into theS. cerevisiae genome at the PDR5 locus of genetically modified strainsresulting in large amounts of the fully functional heterologous membraneprotein being translated, transported to and incorporated into theplasma membrane of S. cerevisiae. However, a different transcriptionsystem regulator may be used to upregulate the expression of a targetheterologous membrane protein as would be appreciated by a skilledworker. For example, the zinc cluster protein Rdr1p is a transcriptionalrepressor of the PDR5 gene and its deletion will therefore result inup-regulating at the PDR5 locus, while other mutations in the PDR1 geneare known to up-regulate expression at the PDR5 locus (Carvajal E, etal, 1997). In addition, alternative constitutive, inducible orcontrollable promoters might be used to control expression from the PDR5locus in place of the PDR5 promoter. These include constitutive S.cerevisiae PDR5 and PMA1 promoters, copper controllable CTR3, glucoseinducible ADH1 and PGK promoters, the galactose inducible GAL promoter,the doxycycline controllable bacterial tet0 promoter (Belli, G. et al,1998) and the tet0::ScHOP1 controllable cassette (Nakayam H., et al, H.et al, 1998).

The mutated transcriptional regulator pdr1-3 is thought to affectexpression of a number of genes that include the PDRE (pleiotrophic drugresponsive element) sequences and to also affect, either positively ornegatively, the expression of some other yeast genes involved inintracellular trafficing of membrane proteins. PDR5 expression appearsto be the most highly up-regulated among genes containing one or moreupstream PDREs. However, it is considered that the coordinatedexpression of multiple genes affected by Pdr1-3p may be required for thefunctional expression of heterologous genes from the PDR5 locus.

The applicability of the present invention is illustrated in theexamples below in which the pdr1-3 mutation is used to drive the stablehigh-level over-expression in S. cerevisiae of functional heterologousmembrane proteins, namely Cdr1p, Cdr2p, Ben^(R)p and Erg11p from thepathogenic fungi Candida albicans and Cdr1p and Pdh1p from Candidagalbrata. Cdr1p, Cdr2p and Pdh1p are membrane proteins of theABC-transporter class related to the S. cerevisiae multidrug efflux pumpPdr5p (Prasad R, et al, 1995). Cdr1p is encoded by the gene most oftenassociated with the fluconazole-resistance of C. albicans clinicalisolates obtained from immunocompromised and debilitated patients(Sanglard D, et al 1995; Sanglard D, et al 1997; White T C, 1997).Ben^(R)p (also referred to as Mdr1p) is a member of the MajorFacilitator Superfamily of membrane transporters that use theelectrochemical gradients of the plasma membrane to transportxenobiotics such as fluconazole (Fling M E, et al, 1991). Althoughconferring drug resistance by transporting fluconazole out of fungalcells, it has a narrower substrate specificity for azole drugs thanCdr1p (Sanglard D, et al, 1995, Sanglard D, et al, 1996). Erg11p is thelanosterol α-14 demethylase of ergosterol metabolism in fungi and is thetarget of fluconazole action (for review see White T C, et al, 1998).Over-expression sufficient to demonstrate drug resistant phenotypes ofconsiderable practical value was achieved by replacing the chromosomalcopy of the PDR5 ORF (open reading frame) with the ORF of C. albicansCDR1 or BEN^(R) or ERG11 in a pdr1-3 mutant depleted in endogenousmembrane transporters. The invention is also illustrated by theproperties of the mutant S. cerevisiae strain which additionallycontains the sec6-4 mutation. The value of such heterologous expressionsystems in studies to determine pump specificity and to screen for pumpantagonists is illustrated. However, other applications of this andderivative systems may be carried out as would be appreciated by aperson skilled in the art. Such potential applications include usesrelated to the heterologous over-expression of P-glycoprotein, thecystic fibrosis transmembrane conductance regulator, and other human,animal, microbial, plant and fungal plasma membrane proteins that can beused in the treatment of disease, and the modulation of physiology,growth or development. The invention may also find application in theover-expression of heterologous membrane proteins for the purposes ofbiochemical or structural analysis of the expressed membrane proteins,enzyme purification and pharmacogenomic applications. A furtherapplication of the system which is contemplated is the use of panels ofisogenic yeast comprising a susceptible control strain plus a set ofconstructs that individually functionally hyper-express molecules thatprovide separate resistant determinants. For example a panel of mutantscomprising the AD1-8u⁻ mutant and derivative strains individuallyhyper-expressing Cdr1p or other ABC-pumps, Ben^(R)p or other MFS pumps,and Erg11p could be used to select for antifungal agents that would notbe susceptible to mechanisms of resistance mediated by these molecules.The panel of strains could be used to select drugs whose intracellulartargets either involved, or did not involve Erg11p, and to identifycompounds whose potency is not compromised by the expression ofmultidrug efflux pumps. This could be applied to either theidentification of new classes of drugs or the refinement of existingclasses of drugs.

In a further embodiment the present invention provides a kit forscreening for drugs useful as a pharmaceutical or agrochemicalcomprising:

-   -   i) a host cell;    -   ii) a vector containing the coding sequence of a target        heterologous membrane protein, said sequence being under the        control of a promoter which, upon transformation of said host        cell and chromosomal integration, causes over-expression of the        functional target protein in the membrane of the host cell; and    -   iii) instructions to carry out said transformation and drug        screening procedures.

The host cell is of the genus Saccharomyces, and most preferably thehost cell is a Saccharomyces cell which has been genetically altered tobe depleted in one or more natural membrane proteins. A suitable hostcell is the Saccharomyces cerevisiae AD1-8u⁻ strain. In anotherembodiment a suitable host cell may be a sec6-4 mutant of the AD1-8u⁻strain. In a further embodiment the host strain may be a derivative ofthe AD1-8u⁻ strain modified to select for a novel phenotype, such asprototrophy, an auxotrophic requirement of drug sensitivity.

The vector may contain elements which allow replication in Escherichiacoli, plus a promoter such as a Sacharomyces cerevisiae promoter andmore preferably the PDR5 promoter. Activity of the Saccharomycespromoter is preferably under the additional control of a mutatedtranscriptional regulator causing over-expression of the target codingsequence, leading to abnormal expression of the target protein in themembrane of the host cell. The mutated transcriptional regulator Pdr1-3pis preferred and is located in the genome of the host cell. In someapplications, a transcriptional terminator that is functional in yeastmay be included in the vector. Either the natural terminator of the geneencoding the membrane protein or the yeast PGK1 terminator is preferred.In other applications immunological, affinity or fluorescent tags may beincluded in the vector. In some further applications, a selectablemarker may also be included in the vector such as S. cerevisiae URA3marker. In other applications a S. cerevisiae centromere or autonomouslyreplicating sequence might be included in the vector. The vector ispreferably pABC3.

Non-limiting examples of the application of this technology, mostparticularly to the understanding of multidrug efflux at the cellularand biochemical level, together with the characterisation of inhibitorsthat are active against prominent fungal pathogens, are set out below.

EXAMPLES Example 1 Production of Plasma Membrane Protein ExpressionSystem Validated by C. albicans Cdr1p Expression in S. cerevisiae

Materials and Methods

Bacterial and Yeast Strains, and Growth Media.

Plasmids were maintained in Escherichia coli DH5α. The CDR1 gene wasobtained from C. albicans ATCC 10261. S. cerevisiae strains used were:AD1-8u⁻ (MATα, pdr1-3, his1, ura3, Δyor1::hisG, Δsnq2::hisG,Δpdr5::hisG, Δpdr10::hisG, Δpdr11::hisG, Δycf1::hisG, Δpdr3::hisG,Δpdr15::hisG, based on AD12345678 [Decottignies, A. et al, 1998]) andAD124567 (MATα, pdr1-3, his1, Δyor1::hisG, Δsnq2::hisG, Δpdr10::hisG,Δpdr11::hisG, Δycf1::hisG, Δpdr3::hisG [Decottignies A, et al, 1998]).E. coli was cultured in LB medium (Sambrook J, et al, 1996). C. albicanswas maintained on YEPD (g/l: yeast extract 10, bacto peptone 20, glucose20), and S. cerevisiae was maintained on YEPD, complete synthetic medium(CSM, Bio 101, Vista, Calif.) or CSM without uracil (CSM-URA, Bio 101)as required.

Plasmid Construction and Yeast Transformation.

Expand DNA polymerase (oche Diagnostics N.Z. Ltd, Auckland, N.Z.) wasused to PCR amplify the CDR1 ORF and transcriptional termination region(4.8 kb) from C. albicans ATCC 10261 genomic DNA using primerscontaining SpeI restriction sites: 5′-CTTTAAAAGGTCAACTAGTAAAAAATTATG-3′and 5′-CAATAATACACTAGTTTGCAACGGAAG-3′. The PCR product was digested withSpeI and cloned into plasmid pSK-PDR5PPUS (FIG. 1A) that had beenpreviously cut with SpeI and dephosphorylated with alkaline phosphatase(New England Biolabs, Beverly, Mass.). The orientation of the CDR1 openreading frame (ORF) was confirmed by sequencing to be the same as PDR5and the plasmid was designated pKEN1002. Plasmid pKEN1002 (FIG. 2A) waslinearized with XbaI and used to transform S. cerevisiae AD1-8u⁻ to Ura⁺by the lithium acetate transformation protocol (Alkali-Cation Yeast kit,Bio-101). The entire CDR1 ORF DNA in pKEN1002 was sequenced, and theCDR1 ORFs from C. albicans ATCC 10261 and S. cerevisiae AD1-8u⁻/pKEN1002transformant AD1002 were PCR amplified from genomic DNA with Pfx.DNApolymerase (Gibco BRL, Life Technologies, Rockville, Md.) and sequenced.

Northern Analysis of RNA Extracted from S. cerevisiae.

Total RNA was extracted from S. cerevisiae as described previously(Albertson G D, et al, 1996). RNA (20 μg) was electrophoresed in agarosegels, vacuum blotted onto Hybond⁺ nylon membrane (Amersham PharmaciaBiotech New Zealand, Auckland, N.Z.) and fixed by UV irradiation.Membranes were hybridized with [α-³²P]dCTP-labeled probes under highstringency conditions as previously described (Cannon R D, et al.,1994). A C. albicans CDR1 probe (ORF nt 1-497) was generated by PCRamplification and the S. cerevisiae PMA1 probe (ORF nt −835-1598) wasobtained as a 2.4 kb BamHI fragment from plasmid pDP100 (Seto-Young, D.et al, 1994).

Immunodetection of C albicans Cdr1p.

Crude protein extracts were prepared from S. cerevisiae cells grown inYEPD broth to mid-exponential phase. Plasma membrane fractions of thesecells were obtained by sucrose gradient centrifugation as previouslydescribed (Monk, B C. et al., 1991). Protein samples (40 μg) wereseparated by electrophoresis in 8% SDS polyacrylamide gels and eitherstained with Coomassie blue or electroblotted (100 V, 1 hour, 4° C.)onto nitrocellulose membranes (Highbond-C, Amersham). Western blots wereincubated with a 1:200 dilution of anti-Cdr1p antibodies (provided by DrD. Sanglard, University Hospital Lausanne, Switzerland).Immunoreactivity was detected using horseradish peroxidase-labeled swineanti-rabbit IgG antibodies at a 1:500 dilution.

Genomic DNA Extraction and Southern Analysis of the C. albicans CDR1Gene Integrated into the S. cerevisiae Genome.

Genomic DNA was prepared from S. cerevisiae cells as describedpreviously (Scherer, S. and Stevens, D A. 1987). Genomic DNA (5 μg) wasdigested with the restriction endonucleases EcoRV, SpeI, BamHI, PstI orEcoRI (NEB), separated in a 0.75% agarose gel, and transferred toHybond⁺ nylon membrane (Amersham). Membranes were hybridized with[α-³²P]dCTP-labeled C. albicans CDR1 probe under high stringencyconditions (Cannon, R D. et al, 1994).

Results

Integration of the C. albicans CDR1 Gene at the PDR5 Locus in S.cerevisiae AD1-8u⁻.

The function of C. albicans Cdr1p was studied with a diminishedbackground of endogenous ABC transporter interference by expressing CDR1in the S. cerevisiae pdr1-3 mutant AD1-8u⁻ that is deleted in 7 majorABC transporters. This was achieved by adapting the pleiotropic drugresistance (PDR) pathway-based membrane protein over-expression system(Decottignies A, et al, 1998) that utilizes the multidrug resistanceregulatory mutation pdr1-3, to up-regulate the PDR5 promoter and resultsin over-expression of the Pdr5p protein in plasma membranes (Balzi E, etal, 1994; Decottignies A, et al, 1994). Hyper-induction of Cdr1p wasachieved by integrating the CDR1 ORF at the S. cerevisiae AD1-8u⁻ PDR5locus downstream from the PDR5 promoter. First, the CDR1 ORF and itstranscription terminator region was PCR amplified from C. albicans ATCC10261 genomic DNA with a high fidelity polymerase and cloned into theSpeI site in plasmid pSK-PDR5PPUS, which is located between the PDR5promoter and PDR5 stop codon (FIG. 1). The resulting plasmid, pKEN1002(FIG. 2) was linearized with XbaI and transformed into S. cerevisiaeAD1-8u⁻ (APDR5: nt 360-1163 deleted) with selection for Ura⁺transformants. This selection protocol was made possible by the presenceof the S. cerevisiae URA3 gene in the PDR5 terminator region ofpKEN1002.

The Ura⁺ S. cerevisiae transformants demonstrated lower sensitivities toazoles than the parental strain, and one (AD1002) was selected forfurther analysis. The doubling time of AD1002 in YEPD and CSM-basedmedia was the same as for the parental strain. To confirm integration ofCDR1 at the PDR5 locus in AD1002, uncut total DNA and restricted genomicDNAs were hybridized with a C. albicans CDR1 probe (FIG. 3). The probe,hybridized with uncut genomic DNA and there was no evidence of anepisomal plasmid. Hybridization of the probe with single bands ofgenomic DNA of expected size after digestion with five separaterestriction endonucleases (EcoRV, 5467 bp; SpeI, 4776 bp; BamHI, 4272bp; PstI, 2236 bp; EcoRI, 1042 bp) indicated that a single integrationevent had occurred at the PDR5 locus. The CDR1 gene from the donorstrain C. albicans ATCC 10261 was sequenced and compared with thesequence from C. albicans strain 1001 (Prasad R, et al, 1995, Genbankaccession number X77589). There were 45 nucleotide differences (over the4503 nt ORF) between the two DNA sequences, but only two amino acidchanges: F427Y and V916I substitutions in ATCC 10261 (see Table 1below). The paucity, and conservative nature, of the amino acidsubstitutions indicates that the CDR1 gene is functionally highlyconserved between strains. The CDR1 gene from plasmid pKEN1002, whichhad been passaged through E. coli, had 21 nucleotide differences fromthe template ATCC 10261 sequence but only 5 amino acid differences:E214Q, S842T, S1021L, E1177K, and A1416E substitutions in pKEN1002. Bycontrast, there were no nucleotide differences between CDR1 amplifiedfrom the S. cerevisiae AD1002 transformant and the CDR1 sequence inplasmid pKEN1002 used in the transformation. This is consistent with thefidelity of the DNA polymerase used to amplify the gene from genomicDNA, and suggests that the differences between the pKEN1002 and C.albicans ATCC 10261 CDR1 sequences comprise allelic differences (seeExample 3), a low frequency of changes caused by PCR and mutations thatwere selected in E. coli. None of the CDR1 sequences (C. albicans 1002,C. albicans ATCC 10261, pKEN1002 or S. cerevisiae AD 1002) contained theCTG codon which is decoded by S. cerevisiae as leucine, but by C.albicans as serine. Substitution of leucine for serine in Cdr1pheterologously expressed in S. cerevisiae AD1002, with consequentialeffects on protein function, is not therefore a problem. TABLE 1Nucleotide and amino acid similarities between CDR1 sequences from C.albicans 1001, C. albicans ATCC 10261, plasmid pKEN1002 and S.cerevisiae AD1002 % CDR1 DNA similarity (N° nucleotide substitutions) C.albicans C. albicans S. cerevisiae 1001 10261 pKEN1002 AD1002 % Cdr1p C.albicans 1001 99.00 (45) 98.62 (62) 98.62 (62) amino acid C. albicans10261 99.87 (2) 99.53 (21) 99.53 (21) similarity pKEN1002 99.53 (7)99.67 (5)   100 (0) (N° amino acid S. cerevisiae AD1002 99.53 (7) 99.67(5)   100 (0) substitutions)Expression of C. albicans CDR1 in S. cerevisiae AD1002.

The expression of C. albicans CDR1 in AD1002 was investigated by aNorthern analysis and by immunodetection of plasma membrane proteins.The expression of PMA1 and CDR1 mRNAs by S. cerevisiae AD1-8u⁻, and bythis strain transformed with pSK-PDR5PPUS or pKEN1002 (AD1002) wasmeasured. PMA1 mRNA, encoding the constitutively expressed plasmamembrane H⁺-ATPase, was expressed in all strains (FIG. 4A). CDR1 mRNAwas expressed only in cells transformed with pKEN1002. Expression ofCdr1p (FIG. 4B) was examined by SDS-PAGE analysis of plasma membraneproteins from these strains and S. cerevisiae AD124567 whichover-expresses Pdr5p (Decottignies A, et al, 1994). No major plasmamembrane protein bands of the size expected for ABC transporters (170kDa [Decottignies A, and A. Goffeau, 1997; Krishnamurthy,. et al, 1998;Prasad R, et al, 1995]) were detected by coomassie blue staining ofsamples from the parental strain AD1-8u⁻. This confirmed the depletionof endogenous pumps in this multiply-deleted strain. In contrast,samples from both AD1002 and AD124567 contained a major protein band at170 kDa which accounted for 10-20% of Coomassie-stained plasma membraneprotein. Only the 170 kDa protein from AD1002 reacted with anti-Cdr1pantibodies (FIG. 4C).

Example 2 Characterisation of a Protein Over-expressed Using the SystemDescribed in Example 1

Materials and Methods

Construction of the AD1-8u⁻ sec6-4 Mutant Strain

Construction of this strain involved the transformation of the AD1-8u⁻strain (containing the wild type SEC6 gene) with a URA3 marked versionof the sec6-4 mutant gene, followed by the selective removal of the URA3marker. In the first step of the procedure the URA3-dp1200 cassette ofplasmid pDDB57 (Wilson et al., Yeast 16:65-70, 2000) was used totemporarily mark the SEC6-4 mutant gene in S. cerevisiae strain SY1(Potenza et al., Yeast 8:549-548, 1992). The URA3-dp1200 cassettecontains C. albicans URA3 and direct repeat sequences of 201 bp flankingthe URA3 marker. This feature allows looping out of the chromosomallyintegrated URA3 gene by homologous recomabination. The cassette alsoincludes 77 bp upstream and 144 bp downstream of the two repeatsequences, respectively. The URA3-dp1200cassette was amplified by PCR asa 1296 bp fragment using the following DNA oligonucleotide primers: +vestrand primer: 5′-TCCCGTCTAGTTAATCACTCGGAAGGAAACAACGAGTGAGGTTTCGTGTCATTCTCTAGATTTTCCCAGTCACGACGTT-3′ and negative strand primer5′TGCTACCAAGCTAACAAAAGGATCAGGCTGCCCAAACGGACGTAGACTCAC TGGGCTCCGTGTGGAATTGTGAGCGGATA-3′. The oligonucleotide sequences homologous to thepDDB57 cassette are underlined. The remaining 60 nucleotides each ofprimer direct the URA3-dp1200 cassette to integrate, at 293 bp to 352 bpfor the +ve strand primer and at 412 bp to 353 bp for the 3′-ve strandprimer, downstream of the TAA stop codon of the SEC6 or SEC6-4 mutantgene in S. cerevisiae. Uracil prototrophs of strain SY1 were selectedafter directed integration of the URA3-dp1200 PCR fragment, viahomologous cross-over, downstream of SY1 sec6-4. The strain designatedSY1::URA3 was verified using PCR using primers flanking the expectedintegration site (+ve strand primer fpS: TCCAGAGAGTATAACTCCTG and −vestrand primer SUB2: TGTTGGAAATTTCTCCCGTG). The SEC6-4-URA3 construct instrain SY1::URA3 was PCR amplified from genomic DNA (+ve strand primerSUB1 AATGCAGGAGTTTTACAGTGGC and −ve strand primer SUB2 as above). TheSUB1 sequence is located immediately 3′ to the upstream ORF and SUB2immediately 5′ to the downstream ORF adjacent to the SEC6 gene,respectively. The resultant 5317 bp PCR fragment, containing the wholeSEC6-4 gene plus the URA3-dp1200 cassette, was purified and used totransform strain AD1-8u⁻ to uracil prototrophy, by replacement of itschromosomal copy of SEC6. The correct directional integration of the PCRfragment, via homologous double cross-over at the SEC6 locus, wasconfirmed by PCR for all uracil prototrophic transformants (using theprimers that verified the construct in strain SY1::URA3). These ura+transformants were then tested for the expected temperature sensitivegrowth phenotype, to verify replacement of SEC6 in AD1-8u⁻ with theSECc6-4 allele of strain SY1. A representative transformant, designatedAD1-8u⁻-sec6-4::URA3, was plated onto CSM agar containing5′-fluoro-orotic acid (5′-FOA) for selective loss of the URA3 marker(Boeke et al., Mol Gen Genet 197:345-346, 1984). Strains that looped outthe URA3 cassette, via a single homologous cross-over between the 201 bpdirect repeat regions, were recovered from these plates. Ura⁻ colonieswere verified using the PCR primer pair fp5/SUB2, in comparison withstrains SY1::URA3, AD1-8u⁻-SEC6::URA3 and AD1-8u⁻-sec6-4::URA3. All theura colonies gave the expected 422 bp PCR fragment which comprises onecopy of the 201 bp direct repeat sequence of the URA3-dp1200 cassette,the 77 bp upstream of the 5′ direct repeat and the 144 bp downstream ofthe 3′ direct repeat. A representative strain was designatedAD1-8u⁻-sec6-4::200.

Minimum Growth Inhibitory Concentration (MIC) Determination.

The MICs of antifungal agents for S. cerevisiae cells were determined bya microdilution test based on the macrodilution reference method of theNational Conmnittee for Clinical Laboratory Standards. Cells (10 μl cellsuspension, 2×10⁵ cells/ml) were inoculated into 90 μl CSM-URA, bufferedwith 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 18 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.0 andcontaining 0.67% (w/v) yeast nitrogen base (YNB) in ricrotitre platewells. For the, uracil-requiring strain AD1-8u⁻ the medium wassupplemented with 0.02% (w/v) uridine. The wells contained, in 200 μl,doubling dilutions of antifungal agents (final concentrations:fluconazole, 40-0.078 μg/ml; itraconazole and ketoconazole, 8-0.016μg/ml). The microtitre plates were incubated at 30° C. for 48 h withshaking and then the growth of cells in individual wells (OD₅₄₀) wasmeasured with a microplate reader (EL 340, Bio-Tek, Winooski, Vt.). TheMIC₈₀ was the lowest concentration of drug that inhibited growth yieldby at least 80% compared to a no-drug control.

Nucleotide Triphosphatase Assays.

Yeast were grown in YEPD, pH 5.5 at 30° C. until they reachedlate-exponential phase of growth (OD_(600 nm)=7), washed twice inice-cold distilled water, and incubated on ice for 30 min to minimizeglucose-stimulated Pma1p activity. Cells were resuspended inhomogenising medium (50 mM Tris pH 7.5, 2 mM EDTA and 1 mMphenylmethylsulfonyl fluoride) and disrupted using a Braun Homogeniser.Cell debris and unbroken cells were removed by centrifuging at 2,000×gat 4° C. for 10 min. A crude membrane fraction was isolated from thecell-free supernatant by centrifuging at 30,000×g at 4° C. for 45 min.Plasma membranes were prepared by centrifugation of the supernatantobtained after selective precipitation of mitochondria at pH 5.2 asdescribed previously (Goffeau, A. and Dufour, J. P., 1988). The plasmamembranes were resuspended in 10 mM Tris pH 7.0, 0.5 mM EDTA and 20%[v/v] glycerol and stored at −80° C. Protein was determined using amicro-Bradford (Bio-Rad Laboratories, Hercules, Calif. [Bradford, M. M.,1976]) assay with gamma-globulin as standard. Nucleotide triphosphataseactivity was measured by incubating the plasma membrane fractions (10μg) at 30° C. in a final volume of 120 μl containing 6 mM NTP, 7 mMMgSO₄ in 59 mM MES-Tris buffer pH 6.0-8.0. To eliminate possiblecontributions from nonspecific phosphatases, vacuolar, or mitochondrialATPases, 0.2 mM ammonium molybdate, 50 mM KNO₃ and 10 mM NaN₃,respectively, were included in assays (Monk B C, et al, 1991). OtherATPase inhibitors (20 μM oligomycin, 20 μM aurovertin B or 100 μMvanadate were added to the reaction where indicated. After 30 min thereaction was stopped by adding 130 μl of a solution containing 1% (w/v)SDS, 0.6 M H₂SO₄, 1.2% (w/v) ammonium molybdate and 1.6% (w/v) ascorbicacid. Inorganic phosphate released from NTPs was measured at 750 nmafter 10 min incubation at room temperature.

Disk Drug Susceptibility Assays.

Drug susceptibility was measured using disk assays on CSM-URA plates(containing 1.5% w/v agar). Plates were seeded with yeast cellssuspended in top agar (5 ml, 10⁵ cells/ml). For the uracil-dependentparental strain, agar was supplemented with 0.02% uridine. Fivemicrolitres of drug solution or solvent control were spotted ontosterile Whatman paper disks on the top agar. The following amounts(nmoles) of drugs were applied to individual disks: fluconazole, 6.5(Pfizer Ltd., Sandwich, Kent, United Kingdom); ketoconazole, 0.094(Janssen Research Foundation, Beerse, Belgium); itraconazole, 0.35(Janssen); miconazole, 0.084 (Janssen); arnphotericin B, 54 (E. R.Squibb & Sons, Princeton, N.J.); rhodamine 6G, 10 (Sigma, Penrose,Auckland, N. Z.); rhodamine 123, 50 (Sigma); trifluoperazine 100(Sigma); benomyl, 10 (Nippon Roche); cycloheximide, S (Sigma); carbonylcyanide p-chlorophenylhydrazone, 490 (CCCP, Sigma); oligomycin, 10(Sigma); nigericin, 100 (Sigma); tamoxifen, 25 (Sigma); naftifine, 50(Novartis), quinidine, 500 (Sigma); valinomycin, 20 (Sigma); verapamil,1000 (Sigma). Agar plates were incubated at 30° C. for 48 h or untilclear growth inhibition zones were visible.

Results

Antifungal Sensitivities of S. cerevisiae Cells Expressing C. albicansCdr1p.

The phenotypic effects on antifungal sensitivity of Cdr1p expression inS. cerevisiae strains with a depleted ABC-transporter background wasmeasured. The parental strain AD1-8u⁻ was exquisitely sensitive tofluconazole, ketoconazole and itraconazole (see Table 2 below).Transformant AD1002 was significantly less sensitive to fluconazole,ketoconazole and itraconazole, with >45-, >31- and >250-fold increasesin MICs, respectively (Table 2). Thus, expression of Cdr1p in thistransformant conferred cross-resistance to different azole antifungaldrugs, as has been shown in other S. cerevisiae strains and in C.albicans (Albertson G D, et al, 1996; Prasad R, et al, 1995). Theseresults together with the SDS-PAGE, Western and Northern analysisindicated that the C. albicans drug resistance gene CDR1 is functionallyover-expressed in S. cerevisiae AD1002. TABLE 2 Antifungal sensitivitiesof S. cerevisiae cells expressing Cdr1p or Cdr2p MIC₈₀ (μg/ml) S.cerevisiae strain Fluconazole Ketoconazole Itraconazole AD1-8u^(−a)0.625 <0.016 <0.016 AD1002^(b) 30 0.5 4 AD1-8/CDR2 64 2 1 AD1-8 sec6-40.5 ND ND AD1-8 sec6-4/CDR1 80 ND ND^(a)Parental strain.^(b)Strain expressing Cdr1p (the MIC values were unaffected bysupplementing the medium with uridine [0.02% w/v]).ND, not determined.These experiments were conducted at 30° C.

In a further set of experiments C. albicans CDR1 was hyper-expressed inAD1-8 sec6-4 and C. albicans CDR2 was hyper-expressed in AD1-8u⁻ (seebelow). Comparison of the isogenic null and hyper-expressing strainsshowed increased resistance to azole drugs due to the high levelexpression of C. albicans CDR2 in the AD1-8 background or CDR1 in theAD1-8u⁻ sec6-4 background (Table 2). These results demonstrate thefunctional expression of Cdr2p in the AD1-8 background and that Cdr1p isfunctional in the sec6-4 derivative of the AD1-8 strain when expressedunder permissive conditions (30° C.). This sec6-4 mutant did not grow at37° C., an ultimately lethal condition that leads to the accumulation ofsecretory vesicles that are unable to fuse with the plasma membrane.

C. albicans Cdr1p-mediated Resistance to a Variety of Drugs.

The sensitivities of the parental S. cerevisiae AD1-8u⁻ strain to avariety of drugs was compared with those of its transformed derivativeAD1002 in order to assess the function of Cdr1p (FIG. 5). Thedifferential sensitivities of these two strains is likely to be due tothe drug efflux driven by Cdr1p. AD1002 showed cross-resistance to allazoles tested, and to the sterol biosynthesis inhibitor naftifine, butnot to the antifungal amphotericin B.

Amphotericin B directly permeabilises the plasma membrane of yeast viaan interaction with ergosterol. As expected, its toxicity in yeast wasnot modified by the over-expression of a multidrug efflux pump.

Transformant AD1002 showed clear resistance to the fluorescent dyesrhodamine 6G and rhodamine 123, with rhodamine 6G showing greatercytotoxicity for the parental S. cerevisiae strain. These dyes have beenreported to be transported by mammalian P-glycoprotein and S. cerevisiaePdr5p and Yor1p (Kolaczkowski M, et al, 1996, Decottingnies A, et al,1998) but PDR5 and YOR1 are deleted in both AD1-8u⁻ and AD1002.Rhodamine 6G and rhodamine 123 are therefore likely to be substrates forCdr1p (Clark F S, et al, 1996; Maesaki S, et al, 1999).

It was further discovered that Cdr1p confers resistance to growthinhibition by the following drugs: the MDR modifier trifluoperazine;protein synthesis inhibitor cycloheximide; ionophoric peptide nigericin;anticancer drug tamoxifen; and calcium channel blocker verapamil. Thestructures and targets of these drugs are diverse and our resultsindicate that Cdr1p has a wide pumping specificity. The drug resistancephenotypes conferred by Cdr1p were similar to those observed in theover-expression of Pdr5p (Kolaczkowski M, et al, 1996). The parentalstrain AD1-8u⁻ was resistant to the remaining drugs: tubulin synthesisinhibitor benomyl; mitochondrial ATPase and Pdr5p inhibitor oligomycin;potassium channel blocker quinidine; and K⁺-selective ionophoriccyclodepsipeptide valinomycin at the concentrations used in this studywhich suggests that these drugs are not substrates of Cdr1p.

Nucleotide Triphosphatase Activity of AD1002.

Plasma membrane fractions from S. cerevisiae AD1002 possessed at leastan order of magnitude higher oligomycin-sensitive ATPase activity thanthe parental strain AD1-8u⁻ over the pH range 6.0-8.0 (FIG. 6). Thisactivity had a pH optimum of around 7.5 and thus was readilydistinguished from the Pma1p ATPase of S. cerevisiae which has a pHoptimum of about 6.0 (Decottignies A, et al, 1994). Furthermore, theactivity of Pma1p is insensitive to oligomycin (Monk B C, et al, 1991)and is specific for ATP (Decottignies A, et al, 1994). C. albicans Cdr1pexpressed in S. cerevisiae AD1002 also showed oligomycin-sensitiveUTPase, CTPase and GTPase activities similar to the ATPase activity, andall these NTPase activities had a slightly alkaline pH optima (see Table3 below). Each NTPase activity of AD1002 was sensitive to 100 μMvanadate, but insensitive to 20 μM aurovertin B. Mitochondrial ATPaseactivity therefore made a negligible contribution to the ATPase activityof these membrane preparations. TABLE 3 Oligomycin-sensitive NTPaseactivities in plasma membrane fractions of parental strain S. cerevisiaeAD1-8u and S. cerevisiae AD1002, expressing Cdr1p Oligomycin-sensitiveNTPase activity (nmole Pi min⁻¹ mg protein⁻¹)^(a) S. cerevisiae UTPaseCTPase GTPase strain pH 6.5 pH 7.5 pH 6.5 pH 7.5 pH 6.5 pH 7.5 AD1-8u 10 1 0 0 0 AD1002 16 30 24 40 27 46^(a)NTPase activities were determined in assays similar to thoseproviding the data presented in FIG. 4. Values represent the differencesin ATPase activities measured in the presence and absence of 20 μMoligomycin. The results are the means of two experiments which did notvary more than 10%.

These results are consistent with the heterologous expression of anucleotide triphosphatase activity with the characteristics expected fora Pdr5p homologue. They provide the first reliable measurement of the invitro activity of Cdr1p. Crd1p is not sufficiently prominent in assayswith C. albicans plasma membranes to be discriminated from theactivities of other ATP utilising enzymes such as the plasma membraneproton pump and endogenous members of the ABC-transporter family. Thenucleotide triphosphatase activity in plasma membranes from AD1002 issufficient to determine several of the biochemical characteristics ofthe enzyme and for it to be used in the screening for, or the assay of,agonists or antagonists that may be candidates for drug discoverypurposes.

Parallel studies indicate that the present invention can be more broadlyapplied. The C. albicans CDR2 gene is a homologue of CDR1. The C.albicans CDR2 ORF was cloned into the pSK-PDR5PPUS vector and used totransform AD1-8u⁻. A resultant Ura⁺ transformant, with the CD A2 ORFintegrated into the PDR5 locus, was resistant to fluconazole (MIC=64μg/ml, Table 2) and was cross-resistant to ketoconazole (2 μg/ml) anditraconazole (1 μg/ml) (Table 2). DNA sequence analysis of PCR productsobtained from the genomic DNA of the transformant showed that the codingregion in the transformed PDR5 locus was identical to that of the CDR2in the genome of the donor strain ATCC 10261. Northern analysis showedthat the CDR2 mRNA was highly expressed in the transformant. Gelelectrophoresis of purified plasma membranes from the transformantrevealed the presence of a major 170 kDa band in amounts comparable tothe endogenous levels of the 100 kDa Pma1p band. In a further study theORFs of the Candida glabrata CDR1 and PDH1 genes, which are related toS. cerevisiae PDR5, were transformed into the PDR5 locus of strainAD1-8u⁻. These constructs, gave genomic DNA sequences in the PDR5 locusthat were identical to the coding sequence of C. glabrata CDR1 and PDH1,respectively. Both constructs expressed the produced expectedheterologous mRNA, hyper-expressed the expected protein product inplasma membrane fractions (identified by internal sequence analysis ofthe proteolytically digested gel band), conferred resistance to azoleand triazole drugs but not polyene antibiotics. Under glucose-energisedconditions, these strains effluxed the ABC-transporter substraterhodamine 6G at high rates while a null mutant failed to effluxrhodamine 6G. Both hyper-expressed proteins could be shown to bephosphorylated in vivo. These results show that the present inventioncan allow high fidelity cloning and confirms the ability to achieveheterologous functional hyper-expression of plasma membrane proteins, inparticular proteins of the ABC-transporter class, in S. cerevisiae.

Example 3 The Heterologous Hyper-expression of Different Classes ofMembrane Protein and its Application to Drug Discovery

Materials and Methods

Preparation of Transformation Cassettes in pABC3

Pfx DNA polymerase (Gibco BRL, Life Technologies, Rockville, Md.) wasused to PCR amplify CDR1, BEN^(R) and ERG11 from C. albicans ATCC 10261genomic DNA and PDR5 from S. cerevisiae AD124567 using primerscontaining PacI or NotI restriction sites: The primers pairs , withrelevant restriction sites underlined, were: 1. CDR1,5′-GTCAAAATTAATTAAAAAATGTCAGATTCTAAGATGTCGTCGCAAG-3′ and5′-CACGCGGCCGCTTAGTGATGGTGATGGTGATGTTTCTTATTTTTTTTCTCTCTGTTACCC-3′ 2.BEN^(R), 5′-CATCTACTTACATTAATTAACACAATGCATTACAG-3′ and5′-GGAAAACAATGCGGCCGCCTAATTAGCATA-3′ 3. ERG11,5′-TTCAAGAAGATTAATTAACAATATGGCTATTGTTGAAACTG-3′ and5′-GAATCGAAAGAAAGCGGCCGCTTTATTAAAACATACAAGTTT-3′, 4. PDR5,5′-GTTTTCGTGGCCGCTCGGGCCAAAGACTTAATTAAAAAATGCCCGAGGC-3′ and5′-ACCCACATATAGCGGCCGCATATGAGAAGACG-3′.

Each PCR product was digested with PacI and NotI and cloned into plasmidpABC3 that had been predigested with PacI and NotI. The orientation ofeach open reading frame (ORF) was confirmed by sequencing to be the sameas PDR5. Each plasmid was digested with AscI and used to transform S.cerevisiae AD1-8u⁻ to Ura⁺ by the lithium acetate transformationprotocol (Alkali-Cation Yeast kit, Bio-101).

Disk Drug Susceptibility Assays.

Drug susceptibility was measured using disk assays on CSM-URA plates(containing 1.5% w/v agar). Yeast cells (200 μl ml of 5×10⁶ cells/ml)were spread on the plates. 10 μl of drug solution or solvent-controlwere spotted onto sterile Whatman paper disks on the pre-spread plates.The following amounts (mnoles) of drugs were applied to individualdisks: fluconazole, 633 (Pfizer Ltd., Sandwich, Kent, United Kingdom);itraconazole, 0.23 (Janssen); miconazole, 0.42 (Janssen); cycloheximide,0.71 (Sigma); rhodamine 6G, 100 (Sigma, Penrose, Auckland, N. Z.);rhodamine 123, 125 (Sigma); cerulenin 4.5 (Sigma); 5-flucytosine 100(Sigma); amphotericin B, 97 (E. R. Squibb & Sons, Princeton, N.J.);nystatin 65 (Sigma). Agar plates were incubated at 30° C. for 48 h oruntil clear growth inhibition zones were visible.

Results

The discovery of a system that functionally hyper-expresses differentclasses of membrane protein in the plasma membrane of S. cerevisiae hasmajor implications for the genetic, physiological, biochemical andstructural study of such molecules. It opens important avenues forpractical application in areas such as drug discovery and biosensing.For example, membrane proteins constitute a high percentage of cellularproteins and they also contribute the major proportion of existing drugtargets, while many biosensors use membrane proteins as receptors or ascomponents of receptor-linked systems in signaling processes. Theprevious two sections of this document showed the functionalheterologous hyper-expression of the C. albicans Cdr1p ABC transporterand noted the hyper-expression of related transporters from C. albicans(Cdr2p) and C. glabrata (Cdr1p and Pdh1p). This was achieved byexpressing these foreign ABC transporter genes, integrated at the PDR5locus in the S. cerevisiae AD1-8u⁻ strain, under control of the pdr1-3gain of function mutation in the Pdr1p transcriptional regulator. Thepresent section provides examples of the broader applicability of thissystem to further classes of membrane proteins. It uses, as anillustrative example, the functional heterologous hyper-expression ofrepresentatives of three distinct classes of membrane proteins from C.albicans that are responsible for determining three separate modes ofresistance to the antifungal drug fluconazole. In particular, itdemonstrates and compares the functional hyper-expression, based on theAD1-8u⁻ host strain, of Ben^(R)p, Erg11p and separate alleles of Cdr1p.The vector used to prepare the transformation cassettes for thesestudies was pABC3 (FIG. 1B). For these studies strain AD1-8u⁻ and strainAD-pABC3 (AD1-8u⁻ transformed with the unmodified transformationcassette from pABC3) were used as negative controls for pump expression(FIG. 7, lanes 7 and 2 respectively). Strain AD-PDR5, prepared with atransformation cassette that included the complete S. cerevisiae PDR5ORF (FIG. 7, lane 3), served as a positive control for pump expression.FIG. 7 shows the Coomassie blue R250-stained protein profiles of plasmamembranes obtained from these control strains and the engineered strainsAD-CDR1 (lane 4), AD-ERG11 (lane 5), and AD-BEN^(R) (lane 6). The lastthree strains were obtained by homologous recombination usingtransformation cassettes containing the C. albicans CDR1, BEN^(R) andERG11 ORFs, respectively. See Materials and methods for the preparationof the transformation cassettes. While AD-BEN^(R) and AD-ERG11 wereconstructed in strain AD1-8u, AD-CDR1 was constructed in the temperaturesensitive strain AD1-8u⁻ sec6-4. As expected, the AD-CDR1 strain wastemperature sensitive when grown at 37° C. When grown at the permissivetemperature of 30° C. plasma membranes from strain AD-CDR1 showed thepresence of a major 170 kDa protein band that was not found in either ofthe two negative control strains. This band was detected in an amountthat was at least 2-fold in excess of the endogenous 100 kDa Pma1p bandand was comparable with the amount of Pdr5p found in membranes fromstrain AD-PDR5, in which the Pma1p and Pdr5p bands were present inequivalent amounts (FIG. 7 compare lanes 3 and 4). We have noted, insome instances, that hyper-expression of ABC-transporters gives up to a50% decrease in the amount of Pma1p found in the plasma membranefraction compared with the control AD1-8u⁻ strain. This decrease is notaccounted for by the increased contribution of the heterologouslyexpressed band to the plasma membrane fraction. Based on theseconsiderations the Cdr1p band contributes at least 10-20% of plasmamembrane protein in strain AD-CDR1. SDS-PAGE analysis of plasmamembranes from the AD-ERG11 construct (FIG. 7 lane 5) showed thepresence of a protein band at about 61 kDa that that was stained toabout half the intensity of Pma1p and was not found in any of thecontrol strains. Assuming equivalent Coomassie blue staining per unitmass of protein, this result indicates the detection of approximatelyequal numbers of Pma1p and Erg11p molecules in plasma membranes fromthis strain. However, the AD-ERG11 membranes gave no indication of anadditional protein band that might correlate with the 680 amino acidPrd1p, the NADPH-cytochrome P-450 reductase that is required for thecatalytic function of Erg11p. The enzyme lanosterol 14α-demethylaseencoded by ERG11 is a member of the family of cytochrome P-450 enzymes.This enzyme is normally located in the endoplasmic reticulum.Comparative cell fractionation studies with strains AD-pABC and AD-ERG11show that while a 61 kDa band specific to the AD-ERG11 strain wasdetectable in microsomal fractions obtained by differentialcentrifugation, the plasma membranes fraction (marked by the 100 kDaPma1p band) showed several-fold higher levels of the 61 kDa band (datanot shown). This indicates that hyper-expression targets excess oflanosterol 14α-demethylase to the plasma membrane, possibly via adefault pathway. Finally, the plasma membrane fraction from theAD-BEN^(R) strain (FIG. 7 lane 6) showed the presence of a slightlyfuzzy protein band at 60 kDa corresponding to the molecular sizeexpected for Ben^(R)p. It was present in amounts approximatelyequivalent to the Pma1p band, was detected immunochemically with anantibody directed against recombinant Ben^(R)p antigen, and was notpresent in the control strains (compare with lanes 2 and 7). Theseresults indicate that the AD-BEN^(R) strain may insert more Ben^(R)pthan Pma1p molecules into the plasma membrane.

Disk diffusion assays, which used the AD-pABC construct as negativecontrol, demonstrated the response of each hyper-expressing construct toindividual toxic antifungal agents and to separate classes of thesemolecules (FIG. 8). Differential resistance/sensitivity profiles wereobtained that reflected the functional hyper-expression and knownspecificity of each recombinant membrane protein. Strains AD-PDR5,AD-CDR1, AD-BEN^(R) and AD-ERG11 showed differential sensitivities tofluconazole that were confirmed by the determination of MICs in liquidmicrodilution assays. Strains AD-PDR5, AD-CDR1, AD-BEN^(R) and AD-ERG11had MICs of 400, 400, 80 and 2 ug/ml fluconazole respectively, comparedwith AD-pABC3 which had an MIC of 0.5 ug/ml fluconazole. AD-PDR5 andAD-CDR1 showed identical resistances to the triazole (fluconazole anditraconazole) and azole (miconazole) drugs, and to cycloheximide,rhodamine 6G and rhodantine 123, as expected for the broad specificityof the ABC-transporters Pdr5p and Cdr1p. The expected sensitivities ofAD-PDR5 and AD-CDR1 to flucytosine (5-FC) and to the polyene antibioticsamphotericin B and nystatin were confirmed in the disk diffusion assays.The compound 5-FC targets the enzyme thymidylate synthase and thepolyene antibiotics target membrane ergosterol, respectively, and arenot known as ABC-transporter substrates. The same assays with AD-BEN^(R)showed that the hyper-expressed Ben^(R)p transporter had narrowersubstrate specificity. Thus, AD-BEN^(R) was resistant to the Ben^(R)psubstrates fluconazole, cycloheximide and cerulenin, it showeddetectable resistance to miconazole, but was fully sensitive to theABC-transporter substrates rhodamine 6G, rhodamine 123 and itraconazole,which are not known as substrates of Ben^(R)p. As expected AD-BEN^(R)was sensitive to 5-FC and the polyene antibiotics. AD-ERG11, in linewith its ability to confer modest resistance to fluconazole, also showedsome resistance to both itraconazole and miconazole, as would beexpected of the hyper-expression of their common target lanosterol14α-demethylase. AD-ERG11 was also susceptible to the drug effluxsubstrates cycloheximide, cerulenin, rhodamine 6G and rhodamine 123, theantifungal 5-FC and both polyene antibiotics tested.

The hyper-expression of Cdr1p, Ben^(R)p and Erg11p in the fluconazolesensitive AD1-8u⁻ background demonstrates unambiguously both functionand specificity for three different classes of membrane proteins whichconfer fluconazole resistance through separate mechanisms.

Many pathogenic fungi, including C. albicans, C. tropicalis, C. krusei(but not C. glabrata), are diploid organisms. The membrane proteinhyper-expression system provides a test for functional differencesbetween alleles because it allows individual alleles to be selectivelyamplified and their phenotypes compared in a defined host deleted ofconfounding background factors. Sequencing of genomic DNA amplified byPCR has identified several single nucleotide polymorphisms (SNPs) in theCDR1 gene of C. albicans strain AD10261 that predict amino aciddifferences between the gene products (data not shown). We havetherefore, as an example, separately hyper-expressed each allele of C.albicans CDR1 in S. cerevisiae AD1-8u⁻. The two 170 kDa Cdr1ps weredetected in equivalent amounts in plasma membrane preparations analysedby SDS-PAGE and the constructs showed differential resistance tofluconazole. Allele 1 (CDR1-1) obtained from strain ATCC10261 appearsidentical to both the published sequence for cloned CDR1 (cloned instrain JG436, Prasad R, et al, 1995) and the sequence available from theC. albicans genome sequencing project (Strain SC5314). Hyper-expressionof CDR1-1 gives an MIC=400 μg/ml for fluconazole. Hyper-expression ofthe second allele (CDR1-2) confers an MIC=80 ug/ml for fluconazole.

The impact of individual gene alleles on fungal disease is poorlyunderstood. The ability to demonstrate clear cut functional differencesbetween alleles differentiated by SNPs, by magnifying their expressionin a minimized background, provides a tool to investigate whether SNPsaffect the evolution of drug resistance through mechanism such as, butnot restricted to, mitotic gene recombination. For example, mutations orother genetic events that cause the high level expression of a more drugresistant allele could render existing drugs like fluconazoleineffective. More generally, the yeast membrane protein hyper-expressionsystem of the invention may be of value in expressing targets, drugprocessing enzymes, or molecules affected by mechanism-based toxicity,such as closely-related or SNP-affected genes encoded by microbialpathogens, fungi, plants, animals or humans. The system could be used toselect for drugs which are fully effective against pathogens or totailor medications which take into account pharmacogenomic differenceswithin individual species or between patients.

The ability to functionally hyper-express individual membrane proteinsin S. cerevisiae can give rise to selectable phenotypes such as drugresistance. This is exemplified by the selection of fluconazoleresistant phenotypes resulting from the expression of versions ofmultidrug efflux pumps from the PDR5 locus in AD1-8u⁻. These phenotypesgive susceptibilities, ranging from <10 μg/ml to 100s of μg/ml offluconazole, and occur for a variety of reasons. These include, but arenot limited to, constructs which restrict expression from the PDR5locus, constructs which have partially or fully compromised functionbecause of mutations in the coding region, or because a partial versionof a foreign gene has been integrated into the PDR5 locus as an interimmeasure designed to circumvent the toxicity that can be associated withthe cloning of genes specifying membrane proteins in E. coli when usingplasmids such as pSK-PDR5PPUS and its derivatives.

The following examples demonstrate that linear DNA sequences can be usedto complement such defects, with the corrected phenotype obtained byselection for a higher level of drug resistance. FIG. 9 shows aconstruct containing an SfiI site immediately upstream of the PacI sitein the pABC3 transformation cassette led to dramatically lowerfunctional expression of Pdr5p. The limitation on expression in thissystem is directly attributed to the presence of the SfiI site and notthe PacI site, because the PacI site alone had no effect on thefunctional expression of Pdr5p from this locus (compare FIG. 9, lanes2-6). The engineered protein expressed from SfiI-containing constructswas barely detectable in SDS-PAGE separated plasma membrane preparationsand the construct showed low fluconazole resistance (MIC<40 μg/ml). Asimilar defect in an AD1-8u⁻ strain engineered to express Cdr1p could becorrected by transformation with a linear DNA PCR fragment encompassing350 nucleotides of the promoter region and 360 nucleotides of the CDR1coding region from plasmid pKEN1002. The PCR product was amplified usingthe primers 5′-ATCACGATTCAGCACCTTT-3′ and 5′-CCCAAAATTTGGCATTGAAA-3′.Transformants were selected on a solid CSM medium that contained 40μg/ml fluconazole plus 2% glycerol and 0.1% glucose as an energysources. This combination of energy sources was used to identify andselect against respiratory incompetent petite yeast that can showenhanced fluconazole resistance. The initial construct in AD1-8u⁻ hadbarely detectable expression of Cdr1p and an MIC for fluconazole of 20μg/ml. The selected respiratory competent construct gave isolates withan MIC for fluconazole of 400 μg/ml and Cdr1p was expressed in plasmamembranes in amounts comparable to that found in AD1002. In anotherexample, successive attempts to clone the PDH1 gene of C. glabrata intothe multicloning site of pSK-PDR5PPUS plasmid failed, possibly due tolethality in E. coli. As an alternative strategy nucleotides 1-530 andnucleotides 4532-5486 of PDH1 were cloned into the HindIII/EcoRI and theXmaI/SpeI sites of pSK-PDR5PPUS vector, respectively, and the resultantKpnI-NotI transformation cassette used to transform the AD1-8u⁻ PDR5locus by homologous recombination, conferring uracil auxotrophy butretaining full sensitivity to fluconazole. A PCR fragment comprisingnucleotides 1-5342 of PDH1 [reference?] was obtained by amplification ofgenomic DNA and used to transform the AD1-8u⁻ derivative host straincontaining the flanking PDH1 fragments to fluconazole resistance(MIC=200 μg/ml). The resultant construct contained a coding regionidentical to that of genomic PDH1 and its 170 kDa protein product wasfunctionally hyper-expressed in the plasma membrane of the S. cerevisiaehost strain (data not shown).

More generally, the ability to select more resistant phenotypes has manyapplications. These include, but are not limited to, the ability tomodulate levels of functional protein expressed from the PDR5 locus, thetransformation of the S. cerevisiae PDR5locus with genes that may bedifficult to clone in E. coli, the creation of chimeric molecules andthe complementation of drug sensitive phenotypes that may be generatedby site-directed mutagenesis and other genetic manipulations. With theability to hyper-express different classes of proteins from thePDR5locus, forms of selection other than the acquisition of fluconazoleresistance are also readily envisaged.

Example 4 Application of Functional Heterologous Hyper-expression ofMembrane Proteins to Drug Discovery

Materials and Methods

Yeast Strains

In addition to the yeast strains described in the previous Materials andmethods section, S. cerevisiae strain AD1234567 (MATα, pdr1-3, his1,Δyor1::hisG, Δsnq2::hisG, Δpdr5::hisG Δpdr10::hisG, Δpdr11::hisG,Δycf1::hisG, Δpdr3::hisG [Decottignies, A. et al, 1998]) was used.

Checkerboard Drug Susceptibility Assays.

Checkerboard drug susceptibility assays were used to measure thechemosensitisation of cells to fluconazole by test compounds such asPdr5p inhibitors. Fluconazole concentration in the CSM-URA medium wasvaried in one dimension (between 0 and 80 μg/ml) and the concentrationof the test compound was varied in the second dimension (between 0 and40 μM). Cell inocula, growth conditions and the optical determination ofgrowth were identical to standard liquid MIC determinations. The assayswere conducted in 6 by 6 well arrays centred in a 96 well microtitreplate using buffered CSM-URA medium at pH 7.0, with fluconazole and/orpeptide included in each well at the indicated concentration. Growthyields were measured after 48 h incubation at 30° C. and all data weretabulated, calculated and displayed using Microsoft EXCEL software.

Fluconazole Accumulation by S. cerevisiae Cells.

The net rate of fluconazole accumulation by early exponential phase S.cerevisiae cells was measured as previously described (Albertson, G. etal, 1996). To examine the energy-dependence of fluconazole accumulation,assays contained 20 mM sodium azide.

Disk Diffusion Assays.

These assays were conducted as described in example 2. Where indicated,disk assays were conducted with agarose in the place of agar. This isrequired to observe effects with peptide inhibitors of Pdr5p, which mayotherwise absorb to agar constituants.

Rhodamine 6G Efflux and the Characterisation of Inhibitors of Cdr1pFunction.

A previously described method (Kolaczkowski, M et al. 1996) was adaptedto measure rhodamine 6G (Sigma) efflux from whole cells. Yeast cellsfrom exponentially growing cultures in YEPD (OD_(600 nm)=0.5) werecollected by centrifugation (3,000×g, 5 min, 20° C.) and washed threetimes with water. The washed cells were resuspended at a concentrationof 0.5×10⁶ to 1.0×10⁷ cells per ml in HEPES-NaOH (50 mM, pH 7.0)containing 5 mM 2-deoxyglucose and 10 μM rhodamine 6G. In someexperiments fluconazole (10 μM) was also added. Cell suspensions wereincubated at 30° C. with shaking for 90 min to allow rhodamineaccumulation under glucose starvation conditions. The starved cells werewashed twice in 50 mM HEPES-NaOH pH 7.0, and portions (400 μl) incubatedat 30° C. for 5 min before addition of glucose (final concentration 2mM) to initiate rhodamine efflux. At specified intervals after theaddition of glucose, cells were removed by centrifugation, andtriplicate 100 μl volumes of the cell supernatants transferred to wellsof 96 well flat-bottom microtitre plates (Nunc, Roskilde, DK). Therhodamine 6G fluorescence of samples was measured using a Cary Eclipsespectrofluorimeter (Varian Inc, Victora Australia). The excitationwavelength was 529 nm (slit 5) and the emission wavelength was 553 nm(slit 10). In some experiments fluconazole at 10 μg/ml was included inall steps of the assays, while in other experiments peptides at theindicated concentration were added to the assay at the beginning of the5 minute incubation at 30° C. prior to the addition of glucose.

Results

The utility of the Cdr1p over-expressing strain AD1002 and other strainsover-expressing membrane proteins for drug discovery purposes isillustrated.

FIG. 10A shows the effect of two purified peptides on oligomycinsensitive Pdr5p ATPase activity. The compound denoted KN20 is a Pdr5pinhibitor with the primary structureD-NH₂-asparagine-tryptophan-tryptophan-lysine-valine-arginine-arginine-arginine-CONH₂.KN20 has a further essential element in the form of a single4-methoxy-2,3,6-trimethylbenzenesulphonyl substituant linked to ofeither one of the tryptophan sidechains, possibly via the nitrogen orthe adjacent carbon {C2} of the indole ring, although other modes ofattachment to the peptide may be possible. KN20 inhibited the oligomycinsensitive ATPase activity of plasma membranes from the AD124567 yeaststrain overexpressing Pdr5p with an 150 of about 3 μM at pH 7.5 (FIG.10A) while the purified non-derivatised peptide was ineffective. KN20also inhibited the oligomycin sensitive ATPase activity of AD1002 plasmamembranes with an I₅₀ of about 8 μM. (FIG. 10B). The inhibition of theCdr1p ATPase at only 2.7-fold higher concentrations than the Pdr5pATPase indicated that KN20 could be developed as a broad-spectruminhibitor of multidrug resistance caused by fungal ABC-transporters.Checkerboard drug susceptibility assays conducted at pH 7.0 demonstratedthat 30 μM KN20 chemosensitised the Pdr5p overexpressing yeast strain toa sub-MIC concentration (80 μM) of fluconazole (FIG. 11A). Disk drugsusceptibility assays (FIG. 11B) visually demonstrated thechemosensitisation of the Pdr5p-overexpressing AD124567 strain to 120μg/ml fluconazole by amounts of peptide which by themselves have littleor no effect on the overall growth of the yeast. In contrast, KN0 (whichis identical to KN20 but lacks the Mtr substituant) was ineffective atchemosensitisation at these concentrations.

AD1002 cells grown in buffered CSM-URA medium at pH 7.0 containing thedrug fluconazole gave a fluconazole MIC of 30 μg/ml (Table 2). In thesame medium but without fluconazole, the cells were completely resistantto 10 μM KN20 and the peptide alone at 20 μM did not affect overallgrowth after 48 h (FIG. 12A). However, exposure to 10 μg/ml fluconazoleplus 10 μM of KN20 completely abolished growth of the strain. Thisresult shows that KN20 synergistically enhances fluconazole inhibitionof growth in strain AD1002. We postulated that KN20 abolishesfluconazole resistance by inhibiting efflux mediated by Cdr1p. Thisinhibition will raise the intracellular concentration of fluconazole tothe extent that ergosterol biosynthesis is inhibited and hence growth isaffected. Disk drug susceptibility assays confirmed that KN20chemosensitises AD1002 to fluconazole at KN20 concentrations that do notaffect overall growth. (FIG. 12B). In contrast, KN0 was ineffective atchemosensitisation at these concentrations. TABLE 4 KN20chemosensitisation of Candida clinical isolates and S. cerevisiaestrains hyper-expressing individual ABC transporters. StrainChemosensitising concentration Fold-sensitisation by KN20 Yeast species(hyper-expressed transporter) KN20 (μM) FCZ (μg/ml) (For FCZ MIC) C.albicans ATCC10261 80 2 64^(a) C. albicans FR2 (Ben^(R)p) 20 8  4 C.glabrata CBS138 40 20  4 C. tropicalis IFO0618 10 10 16^(a) C. kruseiB2399 80 80  2.5 C. dubliniensis CD36 80 0.31 64.5^(a) C. parapsilosis425 0-80 tested 32 tested  1^(b) S. cerevisiae AD12345678u- 7.5 0.125  2AD124567 (Pdr5p) 20 60 10 AD1002 (CaCdr1p) 10 10  4 27A (CaCdr2p) 20 20 4 1B (CgCdr1p) 15 40  8 4 (CgPdh1p) 20 1.25 16 AD-BEN^(R) (Ben^(R)p) 203.75 16 AD-ERG11 (Erg11p) 20 0.25  8^(a)Resistant “tail” eliminated;^(b)No chemosensitisation

KN20 chemosensitisation of AD1002 to fluconazole shows that this straincan be used to select and/or characterise inhibitors of Cdr1p-dependentmultidrug efflux. FIG. 12C and Table 4 shows that KN20 alsochemosensitises the tail of low level resistance seen with populationsof wild type C. albicans, as exemplified by drug susceptibility assaysof strain ATCC 10261. This low level resistance type is commonly seen inC. albicans strains and may be important for the subsequent evolution ofthe intermediate level resistance found in clinical isolates obtainedafter long term prophylactic exposure to fluconazole. KN20 alsochemosensitises the intrinsically fluconazole-resistant Candida glabratastrain CBS 138 and the Candida krusei strain B2399, as well as clinicalisolates of the pathogens Candidia tropicalis, and Candida dubliniensis(Table 4). However it did not chemosensitise C. parapsilosis strain 425.These results suggest that inhibitors like KN20 may find quite broadapplication to the inhibition of multidrug efflux mediated by pumpsrelated to Pdr5p and Cdr1p.

The present invention provides a system to gauge the breadth of actionof inhibitors like KN20, independent of limitations imposed by thegenetic backgrounds of the organisms donating the DNA encoding thetargets. For example, the over-expression of other potential targetssuch as Cdr2p or individual ABC-transporters from other organisms in theAD1-8u⁻ background provides assays to measure the effect of KN20 on thefunctioning of these targets. Table 4 shows that each ABC-transporterconstruct tested was chemosensitised to fluconazole by sub-MICconcentrations of KN20.

The present invention also provides a system to test other aspects ofspecificity for inhibitors like KN20. For example, KN20 might beexpected to chemosensitise fluconazole resistant C. albicans clinicalisolates that specifically over-express Cdr1p but not strains that relyon the over-expression of a Major Facilitator Superfamily TransporterBen^(R)p. However, clinical isolates of C. albicans are geneticallydiverse and the molecular basis of fluconazole resistance is oftenmultifactoral (Albertson et al, 1996). Drug resistance in C. albicanscan involve expression of various combinations Erg11p, Cdr1p andBen^(R)p, as well as unrelated molecules, thereby complicatingunderstanding of the role of specific molecules in the causation ofresistance and in interpreting the effects of specific inhibitors. Theseproblems mean that clinical isolates will often be inappropriateexperimental models for mode of action studies. For example, thefluconazole resistant C. albicans FR2 strain, which has been shown toover-express BEN^(R), was chemosensitised by KN20 (Table 4). Thissuggested but did not prove that KN20 affected a target other than itsABC-transporters. This problem has been overcome by over-expressingfunctional Ben^(R) in strain AD1-8u⁻. The resultant isogenic construct,strain AD-BEN^(R), allows a valid test of the hypothesis thatchemosensitisation by KN20 is directly mediated by inhibition of Cdr1p.In particular, a fluconazole resistant AD-BEN^(R) strain should not bechemosensitised by KN20 or any other specific chemosensitisers of Cdr1p.Conversely, chemosensiters which act directly and specifically onBen^(R)p should not chemosensitise the AD1002 strain or other strainswhich functionally overexpress Cdr1p or Cdr2p in the AD1-8 background.As shown in FIG. 13A and summarized in Table 4, C. albicans Ben^(R)pfunctionally hyper-expressed in strain AD1-8u⁻ is chemosensitised byKN20. This indicates that KN20 has a different target from Cdr1p inwhole cells. KN20 also chemosensitises strain AD-ERG11 to fluconazole(FIG. 13B and Table 4). These data indicate that a target other thanCdr1p may be the primary site of action of KN20 in cell-based assays.The fungal plasma membrane H⁺-ATPase is the primary pump that generatesthe plasma membrane electrochemical gradients which drive the Ben^(R)ppump and the uptake of nutrients needed for the generation of cellularenergy currency (ATP and NADPH) needed by the ABC-transporters andlanosterol 14α-demethylase. The S. cerevisiae plasma membrane H⁺-ATPaseis inhibited by KN20 (I₅₀=10 μM). Concentrations of KN20 of 10 μM orgreater are required to chemosensitise S. cerevisiae cellshyper-expressing Pdr5p, Cdr1p and Ben^(R)p (Table 4). These data and inhouse studies with Pma1p inhibitors provide strong circumstantialevidence that KN20 indirectly chemosensitises multidrug effiux viainhibition of Pma1p.

More generally, a person skilled in the art could apply versions of theMIC, checkerboard and disk drug susceptibility assays to S. cerevisiaestrains over-expressing particular target plasma membrane proteins froma variety of organisms and perhaps enzymatic studies that might beafforded by target over-expression. These assays could be used to screenfor and assess the potency and specificity of agonists or antagonists ofthe target molecule in a way that is not compromised by strainbackgrounds. Such an investigator may also use inhibitors with a definedmode of action to investigate, for example, the nature of the effluxmediated by a particular drug pump.

Fluconazole Accumulation by AD1002.

The accumulation of [³H]fluconazole by S. cerevisiae AD1-8u⁻ and thetransformants AD1002 and AD/pSK-PDR5PPUS was measured (FIG. 14).Energized AD1-8u⁻ or AD/pSK-PDR5PPUS cells accumulated fluconazole overa 15 min timecourse, whereas AD1002 cells did not. Addition of therespiratory chain inhibitor sodium azide to the assay had no effect onthe accumulation of fluconazole by AD1-8u⁻ or AD/pSK-PDR5PPUS cells, butgreatly increased accumulation by AD1002 cells. These results wereconsistent with the multiple drug resistance of AD1002 cells being dueto energy-dependent drug efflux, and indicated that the over-expressedABC-type transporter Cdr1p functioned as expected. An investigatorskilled in the art could develop this method to screen for agonists andantagonists of multidrug efflux and to assess the physiologicalproperties of this process.

C. albicans Cdr1p Mediated Rhodamine Efflux

Over-expression of Cdr1p by strain AD1002 confers the ability to pumpthe fluorescent substrate rhodamine 6G from cells into the medium.Rhodamine 6G has previously been demonstrated to be a substrate of Pdr5pand Yor1p and is dependent on cellular energisation (the provision ofintracellular ATP through glucose fermentation). The AD1002 strain, fromwhich Yor1p has been deleted, can be used to demonstrate competitionwith rhodamine 6G by other Pdr5p substrates such as fluconazole.

As with strain AD1234567 and AD124567 (data not shown),glucose-dependent efflux of rhodamine 6G from S. cerevisiae was notdetectable with strain AD 1-8u⁻ but was readily observed with strainAD1002 (FIG. 15A). The efflux of rhodamine 6G from preloadedde-energised AD1-8u⁻ cells (preloaded by incubation with rhodamine 6Gplus 2-deoxyglucose) did not significantly increase above backgroundlevels in the presence (FIG. 15A) or absence of glucose (data notshown). Efflux from AD1002 cells preloaded with rhodamine 6G requiredthe addition of glucose (FIG. 15A). For AD1002 the extracellularconcentration of rhodamine 6G increased from a background of 30arbitrary units of fluorescence by about 7-fold during 10 minutesfollowing glucose addition and occurred at a rate that was at least30-fold greater than in the presence of 2-deoxyglucose (FIG. 15A). BothAD1-8u⁻ and AD1002 showed similar survival rates following rhodaminepre-treatment, and accumulated equivalent amounts of rhodamine 6G duringpre-treatment in the presence of 2-deoxyglucose, as demonstrated bydetermination of fluorescence released following cell lysis. The Pdr5psubstrate fluconazole inhibited rhodamine efflux from AD1002 cells. Theaddition of fluconazole (10 μM) during preincubation of AD1002 cellswith rhodamine in the presence of 2-deoxyglucose, as well as during allsubsequent steps in the assay gave a 32% reduction in the concentrationof released rhodamine 6G in the 10 min following the addition of glucose(data not shown). In this instance, fluconazole is thought to becompeting directly with rhodamine 6G for efflux via the Pdr5p pump. Thisis consistent with the observation that fluconazole does not affect thein vitro oligomycin activity of the Pdr5p or Cdr1p. FIG. 15B shows thatKN20 inhibited glucose-dependent rhodamine 6G efflux by strain AD124567in a dose-dependent fashion. A 5 minute preincubation with 40 μM KN20resulted in 50% inhibition of rhodamine 6G efflux. These observations,although not discriminating between a direct or indirect effect onPdr5p, demonstrate the principle that the KN20 affects a directlymeasurable aspect of multidrug efflux. The present invention's use ofrhodamine 6G efflux assay could therefore be developed to measure theeffect of inhibitors on Pdr5p and related ABC-transporters.

Discussion

Strategies that seek to determine target specificity or to screen forinhibitory compounds using yeast that over-express a functional targetcan often be complicated by the presence of multiple related endogenousmolecules with various specificities. This problem is particularlyimportant for the study of pumping mechanisms such as those involved inmultidrug efflux. Circumventing this problem by the functionalover-expression of the target in a system that eliminates or minimisesthis undesirable background is a major advantage for structure andfunction studies and in drug discovery. A system for the stable,functional heterologous over-expression of a target membrane protein ina strain of S. cerevisiae depleted in the major drug-efflux pumps:Pdr5p, Yor1p, Snq2p, Ycf1p, Pdr10p, Pdr11p, and Pdr15p has beendemonstrated. Although none of these endogenous pumps is essential, theyconfer on cells overlapping capacities to tolerate xenobiotics(Decottingnies A, et al 1998, Kolaczkowski M, et al, 1996) and cantherefore complicate physiological studies, biochemical analysis and thedrug discovery process.

A specific example concerned the integration of the CDR1 ORF intogenomic DNA. This resulted in the stable inheritance of a single copy ofthe gene at the locus for the S. cerevisiae homologue PDR5. Fusion ofthe CDR1 ORF to the PDR5 promoter in a strain expressing the mutantpdr1-3 transcriptional regulator gives high level over-expression ofCdr1p. This over-expression was demonstrated as increased CDR1 mRNA, andin the appearance of a new 170 kDa protein band accounting for 10-20% ofplasma membrane protein which specifically reacted with anti-C. albicansCdr1p antibodies. The heterologously expressed protein was functional.Its expression conferred on S. cerevisiae multidrug resistance,increased levels of plasma membrane NTPase activity, gave anenergy-dependent reduction in intracellular fluconazole accumulation andenhanced energy-dependent pumping of rhodamine 6G. The drug resistancephenotype was due to the over-expression of Cdr1p and not simply thepdr1-3 mutation, as the latter mutation was also present in thehyper-sensitive parental strain AD1-8u⁻ deleted of seven endogenoustransporters noted above. Related properties have been observed as aresult of the over-expression of C. albicans Cdr2p and C. glabrata Cdr1pand Pdh1p pumps in the AD1-8 background and of C. albicans Cdr1p in asec6-4 derivative of AD1-8. These observations illustrate options forthe broader application of the present invention to other multidrugefflux pumps of the ABC class of transporters (see below).

The high level over-expression of Cdr1p reduced the sensitivity ofAD1-8u⁻ to a variety of structurally unrelated compounds that could bepump substrates. The spectrum of compounds to which Cdr1p conferredresistance was similar to that for Pdr5p (Kolaczkowski M, et al, 1996).The present results demonstrate an effect of Cdr1p expression on drugsensitivity in the absence of seven other major transporters. If theresistance phenotype is mediated by secondary effects on othertransporters, it cannot involve these seven ABC pumps. Thus, we haveprovided clear evidence of rhodamine 6G resistance and efflux mediatedby Cdr1p in the absence of Yor1p.

Plasma membranes from the Cdr1p over-expressing strain AD1002 displayedan oligomycin-sensitive NTPase activity with biochemical properties,including pH activity profiles, similar to Pdr5p—the S. cerevisiaemultidrug efflux pump related to C. albicans Cdr1p (Decottignies A, etal, 1994). The pH optimum for Cdr1p UTPase activity (pH 7.0-8.0) wassignificantly higher than previously reported at pH 6.5 (KrishnamurthyS, et al. 1998) using a plasmid-based expression system. Interestingly,the specific activity of Cdr1p-ATPase was 4-5 times lower than thePdr5p-ATPase activity of the Pdr5p over-expressing strain AD124567measured under the same conditions (unpublished data). Subsequentcloning of both alleles of CDR1 confirm that mutational changes occurredduring the original cloning of the CDR1-2 allele in AD1002 whichaffected enzyme function. Both of the new isolates show NTPaseactivities in vitro that are comparable to hyper-expressed Pdr5p andboth confer significantly higher resistance to fluconazole (MICs of 80and 400 ug/ml compared with 30 μg/ml for AD1002). These observationswith heterologously hyper-expressed CDR1 alleles validate the search forpump antagonists that will not only circumvent the low level trailingtail of fluconazole resistance intrinsic to many wild type strains of C.albicans and the intermediate resistance (<64 μg/ml) seen influconazole-resistant clinical isolates but also overcome the muchhigher levels of resistance that could be encountered with futureisolates.

The heterologous hyper-expression (>10% of plasma membrane protein) of afunctional membrane protein in S. cerevisiae has been shown for bothCdr1p and Cdr2p from C. albicans and Cdr1p and Pdh1p from C. glabrata.This suggests that the invention may be more broadly applied to theheterologous expression of ABC-transporters. The heterologousover-expression of such plasma membrane proteins in a stable manner, inthe types of recipient strain described in this invention, providestargets that can be analysed and utilised in an isogenic background.This will facilitate structure and function studies of individual pumpsand in the development of drugs directed against this class of moleculeswith the requisite specificity for pharmaceutical application. Theheterologous hyper-expression of functional membrane proteins in S.cerevisiae has also been shown for Ben^(R)p and Erg11p from C. albicans,with both molecules being recovered in the plasma membrane in amountscorresponding to at least that for the yeast plasma membrane H⁺-ATPase.Again it is argued that this will facilitate a wide variety of structureand function studies and aid in the development of drugs targeting suchmolecules. Our data provide a precedent for suggesting that many otherclasses of membrane proteins could be functionally hyper-expressed inthis system, providing a practical tool for approaches such as thephysiological, biochemical and structural genomic study of membraneproteins. Since trafficking to the plasma membrane is thought torepresent a default pathway in yeast, a wide variety of hyper-expressedmembrane proteins could be recovered in this organelle, although itcannot be excluded that targeting signals might be sufficient to placehyper-expressed membrane proteins in their normally targeted organelle.

The invention may be used to discover and characterise agents whichchemosensitise cells via their effects on a target protein such a plasmamembrane transporter. The immunosuppressive agent cyclosporine, forexample, which may interact directly with multidrug efflux transporters,potentiates the effect of fluconazole in vitro and in vivo (Marchetti O,et al, 2000; Marchetti O, et al, 2000a). This invention similarly showsthat competition by the Cdr1p substrate fluconazole significantlyreduced the energy-dependent efflux of rhodamine 6G by the Cdr1poverexpressing strain of S. cerevisiae. Furthermore, the lead compoundKN20 has been obtained as a surface-active Pdr5p inhibitor. KN20 wasfound to chemosensitise the Cdr1p overexpressing strain AD1002 tofluconazole in both checkerboard and disk drug susceptibility assays, itinhibited the oligomycin-sensitive ATPase activity of plasma membranesisolated from this strain and inhibited rhodamine 6G efflux in a Pdr5poverexpressing strain in a dose-dependent manner. These results suggestthat KN20 may be a lead broad-spectrum inhibitor of multidrug effluxmediated by ABC-type transporters in pathogenic yeast. The inhibitoralso chemosensitises the fluconazole-resistant CBS 138 strain of Candidaglabrata and the B2399 strain of Candida krusei (Table 4). Although themolecule(s) mediating fluconazole resistance in this C. krusei strainhas yet to be elucidated, fluconazole resistance in CBS 138 appears tobe primarily mediated by the CgCdr1p, a homolog of S. cerevisiae Pdr5pand C. albicans Cdr1p. In addition, KN20 abolishes the low-level tail offluconazole resistance in wild type C. albicans.

KN20 and its congeners are structurally and functionally different fromother previously characterised multidrug efflux inhibitors. Thesubstituted D-octapeptide does not competitively inhibit Pdr5pnucleoside triphosphatase activity because preincubation with ATP didnot modify the response of the enzyme to the inhibitor (data not shown).The inhibitor is therefore unlikely to interact with the catalytic siteof the multidrug efflux pump. The three arginines in KN20 give a highlypositively charged molecule at physiological pH and, based on in-housestudies of related model peptides and the work of others with D-peptides(Mitchell D J et al., 2000), it is unlikely to cross the yeast plasmamembrane. By elimination, KN20 probably directly affects the activity ofPdr5p or Cdr1p by interacting with cell surface features of theseenzymes. While the above arguments may apply to the in vitro action ofKN20, the interaction between KN20 and other surface-exposed plasmamembrane molecules, in particular the Pma1p, indirectly affects thefunction of the multidrug efflux pumps such as Pdr5p and Cdr1p. This wasdemonstrated by showing chemosensitisation of the Ben^(R)p transporterhyper-expressed in the AD1-8u⁻ background at concentrations that affectthe activity of Pma1p. Whatever mechanism is involved, KN20 provides alead for a novel class of inhibitors that may find pharmaceutical oragrochemical application as antifungal chemosensitisers. Our resultsalso imply that surface-active reagents which chemosensitiseABC-transporter mediated multidrug efflux in pathogenic yeast mayrepresent new classes of drugs or drug leads that can be used toincrease the efficacy of antifungal agents that are substrates ofmultidrug efflux. This approach will have application during antifungaltherapy that may be directed against both wild type and resistantclinical isolates of pathogenic fungi or in the study of model yeastsystems. These chemosensitisers may also circumvent the evolution ofresistance e.g. by sensitising survivors in the trailing tail seen insusceptibility testing of wild type yeast. By significantly increasingthe intracellular concentration of antifungal agent, these inhibitorsmay help overcome antifungal resistance mediated not only byABC-transport but also by other mechanisms. These chemosensitisinginhibitors may therefore lengthen the commercial life of existingantifungals, such as fluconazole and other multidrug efflux substrates,by providing more effective formulations and subverting the impact ofresistance. In addition, by making such substrates more potent, it maybe possible to reduce undesirable direct side-effects on the host orminimise deleterious drug interactions.

The high level expression of specific membrane transporters in a S.cerevisiae strain depleted in endogenous pumps opens the possibility ofstudying, both in vivo and in vitro, individual molecules contributingparticular pumping mechanisms and exploiting this knowledge in drugdiscovery. More generally, by increasing the prominence of a particularkind of functional membrane protein in a background deleted of relatedendogenous molecules, structure and function studies that wouldotherwise not be possible can be implemented. The heterologousexpression system will be useful in screening for pump substrates,agonists and antagonists using oligomycin sensitive NTPase activityassays with purified plasma membranes, whole cell chemosensitization(checkerboard and disk drug susceptibility assays), fluconazole uptakeand rhodamine 6G efflux assays. The invention can also be used by thoseskilled in the art to provide, for example, quantitative measures ofchemosensitiser activity in cells and in vitro, which are of value incharacterising and optimising drug candidates. More general applicationsfor other drug targets that can be expressed using adapted forms of theinvention are readily envisaged. One specific example of this would bethe analysis and pharmacogenomic exploitation of closely related genesand genes that contain SNPs. Another specific example would be the useof the system to select for improved antifungals such as azole andtriazole drugs that are not susceptible to drug-resistance mediated bymulti-drug efflux. Furthermore, the network of genes. regulated by thePdr1-3p transcriptional regulator may assist in the functional insertionof ABC-transporters and heterologous proteins into the plasma membraneby providing accessory proteins that are normally used to support Pdr5poverexpression. This facet of the present invention may haveconsiderable advantage over other systems that give high levelexpression without providing the complementary network of moleculesneeded for successful high volume intracellular trafficing andfunctional integration of the heterologous membrane protein into theplasma membrane.

The inclusion of the sec6-4 mutation in the host AD1-8u⁻ background addsa further dimension to the system by allowing new assays that canexploit the orientation and electrochemical properties of secretoryvesicles. It may also allow the hyper-expression of a wide range ofbiologically, pharmaceutically, and agrochemically relevant plasmamembrane proteins for which suitable whole cell and in vitro assays canbe developed by those skilled in the art. This aspect is complemented bythe construction of the pABC3 vector as part of this invention. ThepABC3 vector is designed to simplify the directional cloning of largemembrane proteins such as the ABC-type transporters, avoid the need toconsider down-stream termination sequences and allow ready excision ofthe linear transformation cassette. Other modifications of this vectorthat are envisaged include the provision of vector elements in cassetteform to allow ease of replacement by alternative elements, the insertionof features such as his-tags and other markers that will facilitateprotein purification and studies of subcellular localization, thedevelopment of constructs that will allow plasmid-based expression, andthe modification or replacement of the PDR5 promoter region to allowinducible gene expression. A host strain, such as AD1-8u⁻, could also bemodified to minimize background interference by deleting other hosthomologues of genes to be expressed from the PDR5 or other locus. Apreferred host strain could also be modified to contain a conditionallyactive version of Pdr1-3p for the purposes of inducible expression fromthe PDR5 locus.

It will be appreciated that it is not intended to limit the invention tothe above mentioned examples only, many variations being possible suchas would readily occur to a person of skill in the art without departingfrom the scope of the invention as defined in the accompanying claims.

Industrial Applicability

The present invention provides an in vitro cell based expression systemwhich is useful for high throughput screening for compounds which may beagonists or antagonists of membrane proteins involved in multi-drugresistance.

References

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1. A protein expression system comprising: i) a host yeast cell; and ii)a vector comprising the coding sequence of a target heterologousmembrane protein, said sequence being under the control of a promoterwhich, upon transformation of said host cell and chromosomalintegration, causes over-expression of the functional target protein inthe membrane of the host cell.
 2. A protein expression system as claimedin claim 1, wherein the host cell is a yeast cell of the genusSaccharomyces.
 3. A protein expression system as claimed in claim 1 or2, wherein the host cell comprises a mutant strain deficient in one ormore naturally occurring membrane proteins thereby enabling the targetprotein to be expressed prominently in the membrane of the host cell andto be accessible for drug screening applications.
 4. A proteinexpression system as claimed in claim 3, wherein the host cell isdeficient in drug efflux pump proteins.
 5. A protein expression systemas claimed in any one of claims 2 to 4, wherein the host cell is theSaccharomyces cerevisiae AD1-8u⁻ strain.
 6. A protein expression systemas claimed in any preceding claim, wherein the host cell contains amutation that leads to the formation of secretory vesicles whose abilityto fuse normally with the plasma membrane is temperature sensitive.
 7. Aprotein expression system as claimed in claim 6, wherein the host cellis a sec6-4 mutant of the AD1-8u⁻ strain.
 8. A protein expression systemas claimed in claim 1, wherein the coding sequence of the target proteinis incorporated into the host cell in a defined location in the genome.9. A protein expression system as claimed in claim 1, wherein the codingsequence comprises the entire natural coding sequence of theheterologous target protein, or a functional fragment or variantthereof.
 10. A protein expression system as claimed in claim 8 or 9,where the target heterologous membrane protein is a drug efflux pumpprotein selected from the group consisting of pump proteins involved inmultidrug resistance in fungi, P-glycoprotein, cystic fibrosistransmembrane conductance regulator and any other human, animal, plantor microbial plasma membrane proteins that play a role in conferral ofdrug resistance.
 11. A protein expression system as claimed in any oneof claims 3 to 10 wherein the target heterologous membrane protein is ofthe same class of membrane proteins which have been deleted from thehost cell.
 12. A protein expression system as claimed in claim 11, wherethe target heterologous membrane proteins are selected from Candidaalbicans Cdr1p, Cdr2p and from Candida galbrata Cdr1p and Pdh1p.
 13. Aprotein expression system as claimed in any one of claims 3 to 10wherein the target heterologous membrane protein is of a different classto the class of membrane proteins which have been deleted from the hostcell.
 14. A protein expression system as claimed in claim 13 wherein thetarget heterologous membrane product is selected from the Candidaalbicans Ben^(R)P and Erg11p.
 15. A protein expression system as claimedin claim 1, wherein the vector is a plasmid vector.
 16. A proteinexpression system as claimed in claim 15, wherein the plasmid vectorcontains elements which allow replication in Escherichia coli.
 17. Aprotein expression system as claimed in claim 15, wherein the plasmidvector is pABC.
 18. A protein expression system as claimed in claim 1,wherein the promoter is a Saccharomyces cerevisiae promoter.
 19. Aprotein expression system as claimed in claim 18, wherein the promoteris selected from the group comprising S. cerevisiae PDR5, PMA1, CTR3,ADH1, PGK and GAL and bacterial tet0 promoters and tet0::ScHOP1controllable cassette.
 20. A protein expression system as claimed inclaim 19, wherein the promoter is the PDR5 promoter.
 21. A proteinexpression system as claimed in any one of claims 18 to 20, wherein theSaccharomyces promoter is under the control of a transcriptionalregulator so as to induce over-expression of the target protein codingsequence in the membrane of the host cell.
 22. A protein expressionsystem as claimed in claim 21, wherein the transcriptional regulator isthe Pdr1-3p transcriptional regulator.
 23. A method of screening fordrugs useful as a pharmaceutical or agrochemical comprising the stepsof: i) transforming the chromosomal DNA of a host yeast cell with DNAcomprising the coding sequence of a target heterologous membraneprotein, said sequence being under the control of a host promoterleading to over-expression of the functional target protein in themembrane of the host cell; ii) introducing at least one candidatecompound to said host cell environment or the environment of a plasmamembrane fraction derived from the transformed host strain; and iii)measuring the effect, if any, of the candidate compound on the host cellgrowth and/or viability and/or specific biochemical or physiologicalfunctions mediated by the target membrane protein; and/or measuring thebinding of the candidate compound to the target membrane protein.
 24. Amethod as claimed in claim 23, wherein the host cell is a yeast cell ofthe genus Saccharomyces.
 25. A method as claimed in claim 24, whereinthe host cell is a Saccharomyces cell which has been genetically alteredto be depleted in one or more natural membrane proteins.
 26. A method asclaimed in claim 25, wherein the host cell is the Saccharomycescerevisiae AD 1-8u⁻ strain.
 27. A method as claimed in claim 25 or 26,wherein the host cell is a sec6-4 mutant of the AD1-8u⁻ strain.
 28. Amethod as claimed in claim 23, wherein the target heterologous membraneprotein comprises a drug efflux pump protein.
 29. A method as claimed inclaim 23, wherein the drug efflux drug pump protein is selected from thegroup consisting of pump proteins involved in multidrug resistance infungi, the P-glycoprotein, the cystic fibrosis transmembrane conductanceregulator and other human, animal, plant and microbial plasma membraneproteins that play a role in the conferral of drug resistance.
 30. Amethod as claimed in any one of claims 23 to 29, wherein the targetmembrane protein is a drug efflux pump protein and the candidatecompound is an efflux pump inhibitor.
 31. A method as claimed in claim23, wherein said coding sequence and promoter are introduced into saidhost cell via a plasmid vector.
 32. A method as claimed in claim 31,wherein the plasmid vector contains elements which allow replication inEscherichia coli.
 33. A method as claimed in claim 31, wherein theplasmid vector is pABC3.
 34. A method as claimed in any one of claims 31to 33, wherein said promoter is a Saccharomyces cerevisiae promoter. 35.A method as claimed in claim 34, wherein the promoter is selected fromthe group consisting of S. cerevisiae PDR5, PMA1, CTR3, ADH1, PGK andGAL and bacterial tet0 promoters and the tet0::ScHOP1 controllablecassette.
 36. A method as claimed in claim 34 or 35, wherein thepromoter is the PDR5 promoter.
 37. A method as claimed in any one ofclaims 34 to 36, wherein Saccharomyces promoter is under the control ofa transcriptional regulator so as to induce over-expression of thetarget protein coding sequence in the membranes of the host cell.
 38. Amethod as claimed in claim 37, wherein the transcriptional regulator isthe Pdr1-3p transcriptional regulator.
 39. A vector suitable for use inthe overexpression of a target heterologous membrane protein in a yeasthost cell comprising pABC3.
 40. A bioactive, pharmaceutical oragrochemical compound identified using the method of any one of claims23 to 38 or the protein expression system of any one of claims 1 to 22.41. A compound as claimed in claim 40, wherein said compound wasobtained from compound libraries.
 42. A compound as claimed in claim 40or 41, comprising KN20 as defined herein.
 43. A compound comprising KN20as defined herein suitable for use as an antifungal.
 44. An antifungalcomposition comprising compound KN20 as defined herein together with asuitable carrier or diluent.
 45. An antifungal composition comprisingcompound KN20 as defined herein in a mixture with a known antifungalcompound selected from the group consisting of fluconazole and otherxenobiotics that are transported by multidrug efflux mechanisms.
 46. Apurified membrane protein produced by the method of any one of claims 23to 38 or protein expression system of any one of claims 1 to
 22. 47. Akit for screening for drugs useful as a pharmaceutical or agrochemicalcomprising: (i) a host cell; (ii) a vector containing the codingsequence of a target heterologous membrane protein, said sequence beingunder the control of a promoter which, upon transformation of said hostcell and chromosomal integration, causes over-expression of thefunctional target protein in the membrane of the host cell; and (iii)instructions to carry out said transformation and drug screeningprocedures.
 48. A kit as claimed in claim 47 wherein said host cellcomprises S. crevisiae AD1-8u⁻ and said vector is pABC3 and comprises aPDR5 promoter and a coding sequence of a heterologous drug efflux pumpprotein.
 49. A kit as claimed in claim 47 or 48, wherein said pumpprotein is selected from the group comprising C. albicans Cdr1p, Cdr2p,Ben^(R)p, Erg11p and C. galbrata Cdr1p and Pdh1p.